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Handbook on Sourdough Biotechnology

Marco Gobbetti • Michael Gänzle Editors

Handbook on Sourdough Biotechnology

Editors Marco Gobbetti Department of Soil, Plant and Food Science University of Bari Aldo Moro Bari, Italy

Michael Gänzle Department of Agricultural, Food, and Nutritional Science University of Alberta Edmonton, Canada

ISBN 978-1-4614-5424-3 ISBN 978-1-4614-5425-0 (eBook) DOI 10.1007/978-1-4614-5425-0 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012951618 © Springer Science+Business Media New York 2013 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Contents

1

History and Social Aspects of Sourdough ........................................... Stefan Cappelle, Lacaze Guylaine, M. Gänzle, and M. Gobbetti

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Chemistry of Cereal Grains ................................................................. Peter Koehler and Herbert Wieser

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3 Technology of Baked Goods ................................................................. Maria Ambrogina Pagani, Gabriella Bottega, and Manuela Mariotti

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4 Technology of Sourdough Fermentation and Sourdough Applications ................................................................ Aldo Corsetti

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5 Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria........................................................... Geert Huys, Heide-Marie Daniel, and Luc De Vuyst

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Physiology and Biochemistry of Sourdough Yeasts ........................... M. Elisabetta Guerzoni, Diana I. Serrazanetti, Pamela Vernocchi, and Andrea Gianotti

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Physiology and Biochemistry of Lactic Acid Bacteria ....................... Michael Gänzle and Marco Gobbetti

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Sourdough: A Tool to Improve Bread Structure ................................ Sandra Galle

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Nutritional Aspects of Cereal Fermentation with Lactic Acid Bacteria and Yeast .................................................... Kati Katina and Kaisa Poutanen

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Sourdough and Gluten-Free Products ................................................ Elke K. Arendt and Alice V. Moroni

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Contents

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Sourdough and Cereal Beverages ........................................................ Jussi Loponen and Juhani Sibakov

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Perspectives ........................................................................................... Michael Gänzle and Marco Gobbetti

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Index ...............................................................................................................

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Chapter 1

History and Social Aspects of Sourdough Stefan Cappelle, Lacaze Guylaine, M. Gänzle, and M. Gobbetti

1.1

Sourdough: The Ferment of Life

The history of sourdough and related baked goods follows the entire arc of the development of human civilization, from the beginning of agriculture to the present. Sourdough bread and other sourdough baked goods made from cereals are examples of foods that summarize different types of knowledge, from agricultural practices and technological processes through to cultural heritage. Bread is closely linked to human subsistence and intimately connected to tradition, the practices of civil society and religion. Christian prayer says “Give us this day our daily bread” and the Gospels report that Jesus, breaking bread at the Last Supper, gave it to the Apostles to eat, saying, “This is my body given as a sacrifice for you”. Language also retains expressions that recall the close bond between life and bread: “to earn his bread” and “remove bread from his mouth” are just some of the most common idioms, not to mention the etymology of words in current use: “companion” is derived from cum panis, which means someone with whom you share your bread; “lord”, is derived from the Old English vocabulary hlaford, which translates as guardian of the bread [1]. The symbolic assimilation between bread and life is not just a template that has its heritage in the collective unconscious, but it is probably a precipitate of the history of culture and traditions. Throughout development of the human civilization, (sourdough) bread was preferred over unleavened cereal products, supporting

S. Cappelle (*) • L. Guylaine Puratos Group, Industrialaan 25, Groot-Bijgaarden, Belgium e-mail: [email protected] M. Gänzle Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada M. Gobbetti Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, DOI 10.1007/978-1-4614-5425-0_1, © Springer Science+Business Media New York 2013

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the hypothesis of a precise symbolism between the idea of elaborate and stylish, and that of sourdough. Fermentation and leavening makes bread something different from the raw cereals, i.e. an artifact, in the sense of “made art”. Besides symbolism, sourdough bread has acquired a central social position over time. Bread, and especially sourdough bread, has become central in the diet of peasant societies. This suggests that the rural population empirically perceived sensory and nutritional transformations, which are also implemented through sourdough fermentation. In other words, the eating of bread, and especially of sourdough bread, was often a choice of civilization. The oldest leavened and acidified bread is over 5,000 years old and was discovered in an excavation in Switzerland [2]. The first documented production and consumption of sourdough bread can be traced back to the second millennium B.C. [3]. Egyptians discovered that a mixture of flour and water, left for a bit of time to ferment, increased in volume and, after baking along with other fresh dough, it produced soft and light breads. Much later, microscopic observations of yeast as well as measurements of the acidity of bread from early Egypt demonstrate that the fermentation of bread dough involved yeasts and lactic acid bacteria – the leavening of dough with sourdough had been discovered [4]. Eventually, the environmental contamination of dough was deliberately carried out by starting the fermentation with material from the previous fermentation process. Egyptians also made use of the foam of beer for bread making. At the same time, Egyptians also selected the best variety of wheat flour, adopted innovative tools for making bread, and used high-temperature ovens. The Jewish people learned the art of baking in Egypt. As the Bible says, the Jews fleeing Egypt took with them unleavened dough. In Greece, bread was a food solely for consumption in wealthy homes. Its preparation was reserved for women. Only in a later period, does the literature mention evidence of bakers, perhaps meeting in corporations, which prepared the bread for retail sale. The use of sourdough was adopted from Egypt about 800 B.C. [4]. Greek gastronomy had over 70 varieties of breads, including sweet and savoury types, those made with grains, and different preparation processes. The Greeks used to make votive offerings with flour, cereal grains or toasted breads and cakes mixed with oil and wine. For instance, during the rites dedicated to Dionysus, the god of fertility, but also of euphoria and unbridled passion, the priestesses offered large loaves of bread. The step from the use of sacrificial bread to the use of curative bread was quick. Patients, who visited temples dedicated to Asclepius (the god of medicine and healing), left breads, and, upon leaving the holy place, received a part of the breads back imbued with the healing power attributed to the god [5, 6]. The use of sourdough is also part of the history of North America. The use of sourdough as a leavening agent was essential whenever pioneers or gold prospectors left behind the infrastructure that would provide alternative means of dough leavening. Examples include the Oregon Trail of 1848, the California gold rush of 1849, and the Klondike gold rush in the Yukon Territories, Canada, in 1898. During the 1849 gold rush, San Francisco was invaded by tens of thousands of men and women in the grip of gold fever. Following the gold rush, sourdough bread remained an element that distinguishes the local tradition until today. Some bakeries in San Francisco claim to use sourdough that has been propagated for over 150 years. The predominant

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yeast in San Francisco sourdoughs is not brewer’s yeast but Kazachstania exigua (formerly Saccharomyces exiguus), which is tolerant to more acidic environments. Lactobacillus sanfranciscensis (formerly Lactobacillus brevis subsp. lindneri and sanfrancisco) was first described as a new species in San Francisco sourdough [7]. The use of sourdough during the Klondike gold rush in 1898 resulted in the use of “sourdough” to designate inhabitants of Alaska and the Yukon Territories and is even in use today. The Yukon definition of sourdough is “someone who has seen the Yukon River freeze and thaw”, i.e. a long-term resident of the area. From antiquity to most recent times, the mystery of leavening has also been unveiled from a scientific point of view. The definitive explanation of microbial leavening was given in 1857 by Louis Pasteur. The scientific research also verified an assumption that the Greeks had already advanced: sourdough bread has greater nutritional value. Pliny the elder wrote that it gave strength to the body. The history and social significance of the use of sourdough is further described below for countries such as France, Italy and Germany where this traditional biotechnology is widely used, and where its use is well documented.

1.2

History and Social Aspects of Sourdough in France

The history of sourdough usage in France was linked to socio-cultural and socioeconomic factors. There is little information about sourdough usage and bakery industries (it seems to be more appropriated than baking), in general, in France before the eighteenth century. It seems as if sourdough bread was introduced in Gaul by the Greeks living in Marseille in the fourth century B.C. In 200 B.C., the Gauls removed water from the bread recipe and replaced it with cervoise, a drink based on fermented cereal comparable to beer. They noticed that the cloudier the cervoise, the more the dough leavened. Thus, they started to use the foam of cervoise to leaven the bread dough. The bread obtained was particularly light. During the Middle Ages (400–1400 A.D.), bread making did not progress much and remained a family activity. In the cities, the profession of the baker appeared. The history of bread making in France was mainly linked to Parisian bakers because of the geographic localization of Paris. The regions with the biggest wheat production were near Paris, and Paris had major importance in terms of inhabitants. In that period, the production of bread was exclusively carried out using sourdough fermentation, the only method known at that time. Furthermore, the use of sourdough, thanks to its acidity, permitted baking without salt, an expensive and taxed (Gabelle) raw material, and allowed one to produce breads appropriate for eating habits in the Middle Ages [8]. The seventeenth century marked a turning point in the history of French bakery. Until then, sourdough was used alone to ensure fermentation of the dough even if in some French regions wine, vinegar or rennet was added. Toward 1600 A.D., French bakers rediscovered the use of brewer’s yeast for bread making. The yeast came from Picardie and Flanders in winter and from Paris breweries in summer. The breads

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obtained with this technique were named pain mollet because of the texture of the dough, which was softer than the bread produced up to that point (pain brie). Two French queens, Catherine de Medicis (Henri II’s wife) and Marie de Medicis (Henri IV’s wife) contributed to the success and development of these yeast-fermented breads. In 1666, the use of brewer’s yeast was authorized for bread making but, after a great deal of debate, in 1668, the use of brewers’ yeast was prohibited. Following the request of Louis XIV, the Faculty of Medicine of the Paris University studied the consequences of yeast usage on public health. According to the doctors, yeast was harmful to human health, because of its bitterness, coming from barley and rotting water. Despite this negative conclusion by the Faculty, Parliament, in its decision of 21st March 1670, authorized the use of brewer’s yeast for bread making in combination with sourdough. Besides the apparition of yeast in bread making, during that period, eating habits evolved towards less acidic foods. Thus, back-slopping techniques were adapted in order to reduce bread acidity [9]. The seventeenth century was also a period of development of the French philosophic and encyclopaedic mind and, fortunately, bread making did not escape this movement. Two books detail the art of bread making and provide information on bread-making techniques and knowledge of that period: “L’Art de la Boulangerie” [10] and “Le Parfait Boulanger” [11]. We have already learned that sourdough was obtained from a part of the leavened dough prepared on the day in question. The volume of this dough piece is progressively increased through addition of flour and water (back slopping) to prepare a sourdough that is ready to be used to ferment the dough. The original piece of dough, called levain-chef, must not be too old or too sour. The weight of the levain-chef is doubled or tripled by addition of water and flour leading to the levain de première. After 6 or 7 hours of fermentation, water and flour are added to give the levain de seconde, which is fermented for 4 or 5 hours. Again, water and flour are added. The dough obtained is called levain tout point and after 1 or 2 hours of fermentation is added to the bread dough. This technique called travail sur 3 levains was recommended by Parmentier [11], who imputed the bad quality of Anjou bread to bread making based only on one sourdough. Bread making based on two or three sourdoughs was predominantly used in that period. In addition, it was understood that outside Paris, bread was mainly produced at home by women. It is interesting to note that Malouin had already made the distinction between sourdough and artificial sourdough in 1779 [10]. Artificial sourdough refers to sourdough obtained from a dough that may contain yeast. This distinction between sourdough and artificial sourdoughs remained in the nineteenth century. Until 1840, the yeast was always used in association with sourdough to initiate fermentation. On this date, an Austrian baker introduced a bread-making process in France based on yeast fermentation alone. This technique was called poolish. The bread obtained, called pain viennois, had much success but use of this method remained limited. In the middle of the nineteenth century, bread making based on three sourdoughs progressively disappeared and was replaced by bread making based on two sourdoughs. Indeed, the back slopping, necessary to maintain the fermentative activity of sourdoughs, imposed a hard working rhythm on the bakers. In 1872, the opening of the first factory for the production of yeast from grain fermentation in France by

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Fould-Springer facilitated the development of bread making based on yeast to the detriment of sourdough bread making. This yeast was more active, more constant, with a nice flavour and most of all had a longer shelf life than brewer’s yeast. As a consequence, from 1885, bread making based on polish fermentation was becoming more wide spread. Sourdough bread was, from that time on, called French bread. In 1910, a bill that prohibited night work and, in 1920, the reduction of working hours, necessitated modification within fermentation processes. Sourdough bread making regressed to a greater and greater extent in the cities when bread making based on three sourdoughs totally disappeared even though, in 1914, the first fermentôlevain appeared. After the First World War, the use of yeast was extended from Paris to the provinces. Indeed, yeast that was produced on molasses from 1922 had a better shelf life and was thus easier to distribute over long distances. However, homemade loaves were still produced, even though they no longer existed in the cities, in the country until 1930 in the form of the levain chef, kept in stone jugs, and passed on from one family to another. The return of war in 1939 led to a further reduction in the use of homemade sourdough bread. In 1964, Raymond Calvel [12] wrote that “sourdough bread making does not exist anymore”. Indeed, baker’s yeast was systematically added to promote dough leavening, which permitted one to obtain lighter breads. In addition, the use of baker’s yeast permitted one to better manage bread quality and to reduce quality variations. Two sourdough breadmaking methods remained in this period. The first was a method based on two sourdoughs, which was mainly used in West and South Loire, and the second, more commonly used, method was based on one sourdough with a high level of baker’s yeast. Between 1957 and 1960, the sensory qualities of bread decreased as a consequence of cost reduction. Fermentation time was reduced to a minimum. Sourdough bread was no longer produced. It was only during the 1980s that sourdough bread making gained popularity again thanks to consumer requests for authentic and tasty breads. Since 1990, the availability of starter cultures facilitated the re-introduction of sourdough in bread-making processes. Indeed, these starters permit one to obtain a levain tout-point with a single step and simplify the bread-making process. A regulation issued on 13th September 1993 [13] defined sourdough and sourdough bread. According to Article 4, sourdough is “dough made from wheat or rye, or just one of these, with water added and salt (optional), and which undergoes a naturally acidifying fermentation, whose purpose is to ensure that the dough will rise. The sourdough contains acidifying microbiota made up primarily of lactic bacteria and yeasts. Adding baker’s yeast (Saccharomyces cerevisiae) is allowed when the dough reaches its last phase of kneading, to a maximum amount of 0.2% relative to the weight of flour used up to this point”. This definition allowed one to dehydrate sourdough with the flora remaining active (amounts of bacteria and yeast are indicated). Sourdough can also be obtained by addition of starter to flour and water. Article 3 of the same regulation declares that “Breads sold under the category of pain au levain must be made from a starter as defined by Article 4, just have a potential maximum pH of 4.3 and an acetic acid content of at least 900 ppm”. The syndicat national des fabricants de produits intermédiaires pour boulangerie, patisserie et biscuiterie is working on a new definition of sourdough in order to be closer to the reality of sourdough bread.

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History and Social Aspects of Sourdough in Italy

The people in early Italy mainly cultivated barley, millet, emmer and other grains, which were used for preparation of non-fermented focacce and polenta. Emmer was not only used for making foods, but also performed as a vehicle of transmission in sacred rituals. At first, the Romans mainly consumed roasted or boiled cereals, seasoned with olive oil and combined with vegetables. After contact with Greek civilization, the Romans learned the process of baking and the technique of building bread ovens. Numa Pompilius sanctioned this gastronomic revolution with the introduction of celebrations dedicated to Fornace, the ancient divinity who was the guardian for proper functioning of the bread oven. The Romans gave a great boost to improvements in the techniques of kneading and baking of leavened products, and regulated manufacture and distribution by bakers (pistores). Cato the Elder described many varieties of bread in De agri coltura (160 B.C.), which by then had already spread to Rome: the libum or votive bread, the placenta, a loaf of wheat flour, barley and honey, the erneum, a kind of pandoro, and the mustaceus, bread made with grape must. In the first century A.D., Pliny the Elder [14] refers to several alternative methods of dough leavening, including sourdough that was air-dried after 3 days of fermentation, the use of dried grapes as a starter culture, and particularly the use of back-slopping of dough as the most common method to achieve dough leavening. Pliny the Elder specifically refers to sourdough in his indication that “it is an acid substance carrying out the fermentation”. According to Pliny the Elder, it was generally acknowledged that “consumption of fermented bread improves health” [14]. After the triumph of classical baking, there were no novel developments in this field throughout the Middle Ages. Finding bread and flour in these centuries was difficult, because of involution of agriculture and the famine and epidemics raging at this time. The bread was divided into two categories: black bread, made from flours of different cereals, of little value and reserved for the most humble people, and white bread, made from refined flour, which was more expensive and present on the tables of the rich. A special bread, whose tradition has been preserved to this day in different national or regional varieties, is the Brezel, originating from the South of Germany. It has a characteristic shape of a knotted and dark red crust, which is generated by application of alkali prior to baking, and is sprinkled with coarse salt crystals. According to legend, it was invented by a German court baker in Urach in South West Germany, who, to avoid the loss of his job, was asked by the Duke of Württemberg to develop a bread that allows the sun to shine through three times. This special bread requires 2 days of working: the first to prepare the sourdough with wheat flour, and the second to mix it with water, flour, salt, lard and malt. During the Renaissance, the practice of holding banquets in the courts of the nobles was a triumph for bread, which was presented in various forms in support of the different dishes. In Venice “fugassa” was prepared for the Easter holidays, a sweet bread made with sugar, eggs and butter. In Tuscany, they used to prepare “pane impepato”, while in Milan it appeared as “panettone”. Only towards the end

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of the 1600s was the use of yeast re-introduced for the distribution of luxury bread, which was salty and had added milk. In 1700, a very important innovation in the art of bread making was disseminated: the millstones in mills were replaced with a series of steel rollers. This allowed cheaper refining of flour. Also, pioneering mixers were set up. With the advances brought by the industrial revolution, bread was increasingly emerging as a staple food for workers. Rather than making the bread at home, people preferred to buy it from bakers. This change was criticized as distorting traditional values. At the same time, a health movement that originated in America started a battle against leavened bread, stating it was deleterious to health. Baker’s yeast was considered a toxic element, perhaps because it was derived from beer, while the sourdough gave a bad taste to the bread, which was remediated by the addition of potash, equally harmful. When Louis Pasteur discovered that microorganisms caused the fermentation, the concern over the toxicity of biological agents was amplified. Pasteur’s discovery eventually benefitted the supporters of the bread, as they stated that the use of selected yeast and related techniques was helpful in the manufacture of bread with a longer shelf life. The education of taste in different food cultures explains, however, the different relationship that has existed between the perception of the quality of bread and its level of acidity. During the First World War, the so-called “military bread” was used in Europe, which was a loaf of 700 g weight with a hard crust. It was initially distributed to soldiers and then also passed on to the civilian population. In the post-war period, thanks to the much-discussed Battle of Wheat, strongly supported by Mussolini, the production of wheat was plentiful and the bread was brought to the table of the general population. The Second World War again resulted in an insufficient supply of bread. With the arrival of the American allies, the bread of liberation – a square white bread – became disseminated. Today, bread is regaining some importance. With a turnaround in the culinary habits of Westerners, bread made with unrefined flour, so-called black bread, is more widely consumed. A brief mention should be made, finally, of the various breads that are currently made with modern baking practices. Typical breads, with PDO (Denomination of Protected Origin) or PGI (Protected Geographical Indication) status, are the Altamura bread, the bread of Dittaino, the Coppia Ferrasese, the bread of Genzano and the Cornetto of Matera. The manufacture of these breads is based on new processes, but still at an artisanal level [15].

1.4

History and Social Aspects of Sourdough in Germany

Acidified and leavened bread has been consistently produced in Central Europe (contemporary Austria, Germany, and Switzerland) for over 5,000 years. Leavened and acidified bread dating from 3,600 B.C. was excavated near Bern, Switzerland [2]; comparable findings of bread or acidified flat bread were made in Austria (dating from 1800 B.C.) and Quedlinburg, Germany (dating from 800 B.C.) [16]. It remains unknown whether these breads represent temporary and local traditions or a permanent

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and widespread production of leavened and acidified bread; however, these archaeological findings indicate that the use of sourdough for production of leavened breads developed independently in Central Europe and the Mediterranean. Paralleling the use of leavening agents in France, sourdough was used as the sole leavening agent in Germany until the use of brewer’s yeast became common in the fifteenth and sixteenth centuries [4, 16]. In many medieval monasteries, brewing and baking were carried out in the same facility to employ the heat of the baking ovens to dry the malt, and to use the spent brewer’s yeast to leaven the dough. The close connection between brewing and baking is also documented in the medieval guilds. In Germany, bakers and brewers were often organized in the same guild. In many cities, bakers also enjoyed the right to brew beer [17]. Baker’s yeast has been produced for use as a leavening agent in baking since the second half of the nineteenth century [4, 16, 18]. Baker’s yeast was initially produced with cereal substrates, but the shortage of grains in Germany in the First World War forced the use of molasses as a substrate for baker’s yeast production [4]. Although artisanal bread production relied on the use of sourdough as the main leavening agent until the twentieth century, the use of baker’s yeast widely replaced sourdough as the leavening agent. Maurizio indicates in 1917 that baker’s yeast was the predominant leavening agent for white wheat bread, whereas whole grain and rye products continued to be leavened with sourdough [19]. In 1954, Neuman and Pelshenke referred to baker’s yeast as the main or sole leavening agent for wheat bread and as an alternative leavening agent in rye bread [20]. The industrial production of baker’s yeast to achieve leavening in straight dough processes was followed by the commercial production of sourdough starter cultures in Germany from 1910. The continued use of sourdough in Germany throughout the twentieth century particularly relates to the use of rye flour in bread production. Rye flour requires acidification to achieve optimal bread quality. Acidification inhibits amylase activity and prevents starch degradation during baking. Moreover, the solubilisation of pentosans during sourdough fermentation improves water binding and gas retention in the dough stage. Following the introduction of baker’s yeast as a leavening agent, the aim of sourdough fermentation in rye baking shifted from its use as a leavening agent to its use as an acidifying agent [18]. This use of sourdough for acidification of rye dough in Germany is paralleled in other countries where rye bread has a major share of the bread market, including Sweden, Finland, the Baltic countries, and Russia. For example, the industrialization of bread production in the Soviet Union in the 1920s led to the development of fermentation equipment for the large scale and partially automated production of rye sourdough bread [21]. Chemical acidulants for the purpose of dough acidification became commercially available in the twentieth century as alternatives to sourdough fermentation. However, artisanal as well as industrial bakeries continued to use sourdough fermentation owing to the substantial difference in product quality. To differentiate between chemical and the more labour-intensive and expensive biological acidification, German food law provided a definition of sourdough as dough containing viable and metabolically active lactic acid bacteria, and defines sourdough

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bread as bread where acidity is exclusively derived from biological acidification. Sourdough is thus one of very few intermediates of food production that is regulated by legislation, and recognized by many consumers [4]. The consumer perception as well as the regulatory protection of the term “sourdough” in Germany and other European countries facilitated the recent renaissance of sourdough use in baking. In comparison, the term “sourdough” is not protected in the United States and the widespread labelling of chemically acidified bread as “sourdough bread” resulted in a widespread consumer perception of sourdough bread as highly acidic bread, and the use of alternative terminology to label bread produced with biological acidification. The commercialization of dried sourdough with high titratable acidity constituted a compromise between economic bread production based on convenient use of baking improvers, and the use of sourdough fermentation for improved bread quality. These products were introduced in the 1970s [18]. Their economic importance rapidly surpassed the importance of sourdough starter cultures. Dried or stabilized sourdoughs produced for acidification provided the conceptual template for the increased use of sourdough products as baking improvers over the last 20 years. Sourdough fermentation was thus no longer confined to small-scale, artisanal fermentation to achieve dough leavening and/or acidification. Sourdough fermentation is also carried out in industrial bakeries at a large scale matching large-scale bread production, and in specialized ingredient companies for production of baking improvers specifically aimed at influencing the storage life as well as the sensory and nutritional quality of bread.

References 1. Mc Gee H (1989) Il cibo e la cucina. Scienza e cultura degli alimenti. Muzzio, Padova 2. Währen M (2000) Gesammelte Aufsätze und Studien zur Brot- und Gebäckkunde und –geschichte. In: Eiselen H (ed) Deutsches Brotmuseum Ulm, Germany 3. Adrrario C (2002) “Ta” Getreide und Brot im alten Ägypten. Deutsches Brotmus eum, Ulm 4. Brandt MJ (2005) Geschichte des Sauerteiges. In: Brandt MJ, Gänzle MG (eds) Handbuch Sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 1–5 5. Moiraghi C (2002) Breve storia del pane. Lions Club Milano Ambrosiano, Milano 6. Guidotti MC (2005) L’alimentazione nell’antico Egitto, in Cibi e sapèori nel Mondo antico. Sillabe, Livorno, pp 18–24 7. Kline L, Sigihara RF (1971) Microorganisms of the San Fransisco sour dough bread process. II. Isolation and characterization of undescribed bacterial species responsible for the souring activity. Appl Microbiol 21:459–465 8. Roussel P, Chiron H (2002) Les pains français: évolution, qualité, production, Sciences et Technologie des Métiers de Bouche. Maé-Erti, Vezoul 9. Dewalque Marc, La lecture du levain au XVIIIième siècle sur http://www.boulangerie.net/ forums/bnweb/dt/lecturelevain/lecturelevainacc.php, consultée le 07/06/2012 à 14h42 10. Malouin PJ (1779) L’Art de la boulangerie ou La description de toutes les méthodes de pétrir, pour fabriquer les différentes sortes de pastes et de pains, 2nd edn. Paris 11. Parmentier AA (1778) Le parfait boulanger ou Traité complet sur la fabrication & le commerce du pain. Imprimerie royale, Paris

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12. Calvel R (1964) Le pain et la panification. Que sais-je ? Presses universitaires de France, Paris 13. Décret n°93-1074 du 13 septembre 1993 pris pour l’application de la loi du 1er août 1905 en ce qui concerne certaines catégories de pains 14. Pliny the Elder G (1972) Naturalis Historia XVIII, 102–104, edition of Le Biniec H; Pline L’Ancien, Historie Naturelle, Livre XVIII, Societé D’Editions le Belles Lettres, Paris 15. Buonassisi V (1981) Storia del pane e del forno. SIDALM, Milano 16. Spicher G, Stephan H (1982) Handbuch Sauerteig, 1st edn. Behr’s Verlag, Hamburg 17. Krauß I (1994) Heute back’ ich, morgen brau’ ich. Eiselen Stiftung Ulm, Ulm 18. Brandt MJ (2007) Sourdough products for convenient use in baking. Food Microbiol 24:161–164 19. Maurizio A (1917) Die Nahrumgsmittel aus Getreide. Parey, Berlin 20. Neumann MP, Pelshenke PF (1954) Brotgetreide und Brot, 5th edn. Parey, Berlin 21. Böcker G (2006) Grundsätze von Anlagen für Sauerteig. In: Brandt MJ, Gänzle MG (eds) Handbuch sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 329–352

Chapter 2

Chemistry of Cereal Grains Peter Koehler and Herbert Wieser

2.1

Introductory Remarks

Cereals are the most important staple foods for mankind worldwide and represent the main constituent of animal feed. Most recently, cereals have been additionally used for energy production, for example by fermentation yielding biogas or bioethanol. The major cereals are wheat, corn, rice, barley, sorghum, millet, oats, and rye. They are grown on nearly 60% of the cultivated land in the world. Wheat, corn, and rice take up the greatest part of the land cultivated by cereals and produce the largest quantities of cereal grains (Table 2.1) [1]. Botanically, cereals are grasses and belong to the monocot family Poaceae. Wheat, rye, and barley are closely related as members of the subfamily Pooideae and the tribus Triticeae. Oats are a distant relative of the Triticeae within the subfamily Pooideae, whereas rice, corn, sorghum, and millet show separate evolutionary lines. Cultivated wheat comprises five species: the hexaploid common (bread) wheat and spelt wheat (genome AABBDD), the tetraploid durum wheat and emmer (AABB), and the diploid einkorn (AA). Triticale is a man-made hybrid of durum wheat and rye (AABBRR). Within each cereal species numerous varieties exist produced by breeding in order to optimize agronomical, technological, and nutritional properties. The farming of all cereals is, in principle, similar. They are annual plants and consequently, one planting yields one harvest. The demands on climate, however, are different. “Warm-season” cereals (corn, rice, sorghum, millet) are grown in tropical lowlands throughout the year and in temperate climates during the frostfree season. Rice is mainly grown in flooded fields, and sorghum and millet are adapted to arid conditions. “Cool-season” cereals (wheat, rye, barley, and oats) grow best in a moderate climate. Wheat, rye, and barley can be differentiated into

P. Koehler (*) • H. Wieser German Research Center for Food Chemistry, Lise-Meitner-Strasse 34, 85354 Freising, Germany e-mail: [email protected] M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, DOI 10.1007/978-1-4614-5425-0_2, © Springer Science+Business Media New York 2013

11

P. Koehler and H. Wieser

12 Table 2.1 Cereal production in 2010 [1] Cultivated area Species (million ha) Corn 162 Rice 154 Wheat 217 Barley 48 Sorghum + millet 76 Oats 9 Triticale 4 Rye 5

Grain production (million tons) 844 672 651 123 85 20 13 12

winter or spring varieties. The winter type requires vernalization by low temperatures; it is sown in autumn and matures in early summer. Spring cereals are sensitive to frost temperatures and are sown in springtime and mature in midsummer; they require more irrigation and give lower yields than winter cereals. Cereals produce dry, one-seeded fruits, called the “kernel” or “grain”, in the form of a caryopsis, in which the fruit coat (pericarp) is strongly bound to the seed coat (testa). Grain size and weight vary widely from rather big corn grains (~350 mg) to small millet grains (~9 mg). The anatomy of cereal grains is fairly uniform: fruit and seed coats (bran) enclose the germ and the endosperm, the latter consisting of the starchy endosperm and the aleurone layer. In oats, barley, and rice the husk is fused together with the fruit coat and cannot be simply removed by threshing as can be done with common wheat and rye (naked cereals). The chemical composition of cereal grains (moisture 11–14%) is characterized by the high content of carbohydrates (Table 2.2) [2, 3]. Available carbohydrates, mainly starch deposited in the endosperm, amount to 56–74% and fiber, mainly located in the bran, to 2–13%. The second important group of constituents is the proteins which fall within an average range of about 8–11%. With the exception of oats (~7%), cereal lipids belong to the minor constituents (2–4%) along with minerals (1–3%). The relatively high content of B-vitamins is, in particular, of nutritional relevance. With respect to structures and quantities of chemical constituents, notable differences exist between cereals and even between species and varieties within each cereal. These differences strongly affect the quality of products made from cereal grains. Because of the importance of the constituents, in the following we provide an insight into the detailed chemical composition of cereal grains including carbohydrates, proteins, lipids, and the minor components (minerals and vitamins).

2.2

Carbohydrates

Cereal grains contain 66–76% carbohydrates (Table 2.2), thus, this is by far the most abundant group of constituents. The major carbohydrate is starch (55–70%) followed by minor constituents such as arabinoxylans (1.5–8%), b-glucans (0.5– 7%), sugars (~3%), cellulose (~2.5%), and glucofructans (~1%).

13

2 Chemistry of Cereal Grains Table 2.2 Chemical composition of cereal grains (average values) [2, 3] Wheat Rye Corn Barley Oats (g/100 g) Moisture 12.6 13.6 11.3 12.1 13.1 Protein (N × 6.25) 11.3 9.4 8.8 11.1 10.8 Lipids 1.8 1.7 3.8 2.1 7.2 Available carbohydrates 59.4 60.3 65.0 62.7 56.2 Fiber 13.2 13.1 9.8 9.7 9.8 Minerals 1.7 1.9 1.3 2.3 2.9 (mg/kg) Vitamin B1 (thiamine) 4.6 3.7 3.6 4.3 6.7 Vitamin B2 (riboflavin) 0.9 1.7 2.0 1.8 1.7 Nicotinamide 51.0 18.0 15.0 48.0 24.0 Panthothenic acid 12.0 15.0 6.5 6.8 7.1 Vitamin B6 2.7 2.3 4.0 5.6 9.6 Folic acid 0.9 1.4 0.3 0.7 0.3 Total tocopherols 41.0 40.0 66.0 22.0 18.0

2.2.1

Rice

Millet

13.0 7.7 2.2 73.7 2.2 1.2

12.0 10.5 3.9 68.2 3.8 1.6

4.1 0.9 52.0 17.0 2.8 0.2 19.0

4.3 1.1 18.0 14.0 5.2 0.4 40.0

Starch

Starch is the major storage carbohydrate of cereals and an important part of our nutrition. Because of its unique properties starch is important for the textural properties of many foods, in particular bread and other baked goods. Finally, starch is nowadays also an important feedstock for bioethanol or biogas production (for reviews see [4, 5]).

2.2.1.1 Amylose and Amylopectin Starch occurs only in the endosperm and is present in granular form. It consists of the two water-insoluble homoglucans amylose and amylopectin. Cereal starches are typically composed of 25–28% amylose and 72–75% amylopectin [6]. Mutant genotypes may have an altered amylose/amylopectin ratio. “Waxy” cultivars have a very high amylopectin level (up to 100%), whereas “high amylose” or “amylostarch” cultivars may contain up to 70% amylose. This altered ratio of amylose/amylopectin affects the technological properties of these cultivars [7, 8]. High-amylose wheat has been suggested as a raw material for the production of enzyme-resistant starch [9]. Amylose consists of a-(1,4)-linked d-glucopyranosyl units and is almost linear. Parts of the molecules also have a-(1,6)-linkages providing slightly branched structures [10, 11]. The degree of polymerization ranges from 500 to 6,000 glucose units giving a molecular weight (MW) of 8 × 104 to 106. Amylopectin is responsible for the granular nature of starch. It contains 30,000–3,000,000 glucose units and, therefore, it has a considerably higher MW (107–109) than amylose [12]. Amylopectin is a highly branched polysaccharide consisting of a-(1,4)-linked d-glucopyranosyl

P. Koehler and H. Wieser

14

chains, which are interconnected via a-(1,6)-glycosidic linkages, also called branch points [13]. The a-(1,4)-linked chains have variable length of 6 to more than 100 glucose units depending on the molecular site at which they are located. The unbranched A- or outer chains can be distinguished from the branched B- or inner chains, which can be subdivided into B1-, B2-, B3-, and B4-chains [14]. The molecules are “terminated” by a single C-chain containing the reducing glucose residue [15]. Amylopectin has a tree-like structure, in which clusters of chains occur at regular intervals along the axis of the molecule [16]. Short A- and B1-chains of 12–15 glucose residues form the clusters which have double-helical structures. The longer, less abundant B2-, B3-, and B4-chains interconnect 2, 3 or 4 clusters, respectively. B2-chains contain approximately 35–40, B3-chains 70–80, and B4-chains up to more than 100 glucose residues [12, 17].

2.2.1.2

Starch Granules

In the endosperm starch is present as intracellular granules of different sizes and shapes, depending on the cereal species. In contrast to most plant starches, wheat, rye, and barley starches usually have two granule populations differing in size. Small spherical B-granules with an average size of 5 mm can be distinguished from large ellipsoid A-granules with mean diameters around 20 mm [18]. In the polarization microscope native starch granules are birefringent indicating that ordered, partially crystalline structures are present in the granule. The degree of crystallinity ranges from 20 to 40% [19] and is primarily caused by the structural features of amylopectin. It is thought that the macromolecules are oriented perpendicularly to the granule surface [12, 16] with the nonreducing ends of the molecules pointing to the surface. A model of starch granule organization from the microscopic to the nanoscopic level has been suggested [12]. At the microscopic level alternating concentric “growth rings” with periodicities of several hundreds of nanometers can be observed. They reflect alternating semicrystalline and amorphous shells [12]. The latter are less dense, enriched in amylose, and contain noncrystalline amylopectin. They further consist of alternating amorphous and crystalline lamellae of about 9–10 nm [20]. Crystalline regions contain amylopectin double helices of A- and B1-chains oriented in parallel fashion and possibly 18 nm-wide, left-handed superhelices formed from double helices. Amorphous regions represent the amylopectin branching sites, which may also contain a few amylose molecules. The lamellae are organized into larger spherical blocklets, which vary periodically in diameter between 20 and 500 nm [21]. The amylopectin double helices may be packed into different crystal types. The very densely packed A-type is found in most cereal starches, while the more hydrated tube-like B-type is found in some tuber starches, high amylose cereal starches, and retrograded starch [12, 19]. Mixtures of A- and B-types are designated C-type.

2 Chemistry of Cereal Grains

2.2.1.3

15

Changes in Starch Structure During Processing

In many cereal manufacturing processes flour and also starch is usually dispersed in water and finally heated. In particular heating induces a series of structural changes. This process has been termed gelatinization [22]. Depending on water content, water distribution, and intensity of heat treatment the molecular order of the starch granules can be completely transformed from the semicrystalline to an amorphous state. The mixing of starch and excess water at room temperature leads to a starch suspension. During mixing starch absorbs water up to 50% of its dry weight (1) because of physical immobilization of water in the void space between the granules, and (2) because of water uptake due to swelling. The latter process increases with temperature. If the temperature is below the gelatinization temperature, the described changes are reversible. As the temperature increases, more water permeates into the starch granules and initiates hydration reactions. Firstly, the amorphous regions are hydrated thereby increasing molecular mobility. This also affects the crystalline regions, in which amylopectin double helices dissociate and the crystallites melt [23, 24]. These reactions are endothermic and irreversible. They are accompanied by the loss of birefringence, which can be observed under the polarization microscope. Endothermic melting of crystallites can also be followed by differential scanning calorimetry (DSC). Viscosity measurements, for example in an amylograph or a rapid visco analyzer, also allow one to monitor the gelatinization process. Characteristic points are the onset temperature (To; ca. 45 °C), which reflects the initiation of the process, as well as the peak (Tp; ca. 60 °C) and conclusion (Tc; ca. 75 °C) temperatures. These temperatures are subject to change depending on the botanical source of the starch and the water content of the suspension. The loss of molecular order and crystallinity during gelatinization is accompanied by further granule swelling due to increased water uptake and a limited starch solubilization. Mainly amylose is dissolved in water, which strongly increases the viscosity of the starch suspension. This phenomenon has been termed “amylose leaching,” and it is caused by a phase separation between amylose and amylopectin, which are immiscible [25]. During further heating beyond the conclusion temperature of gelatinization swelling and leaching continue and a starch paste consisting of solubilized amylose and swollen, amorphous starch granules is formed. The shapes of the starch granules can still be observed unless shear force or higher temperatures are applied [23, 26]. Upon cooling with mixing the viscosity of a starch paste increases, whereas a starch gel is formed on cooling without mixing at concentrations above 6%. The second process is relevant in cereal baked goods. The changes that occur during cooling and storage of a starch paste have been summarized as “retrogradation” [22]. Generally, the amorphous system reassociates to a more ordered, crystalline state. Retrogradation processes can be divided into two subprocesses. The first is related to amylose and occurs in a time range of minutes to hours, the second is caused by amylopectin and takes place within hours or days. Therefore, amylose retrogradation is responsible for the initial hardness of a starch gel or bread, whereas amylopectin retrogradation determines the long-term gel structure, crystallinity, and hardness of a starch-containing food [27].

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On cooling granule remnants that are enriched in amorphous amylopectin become incorporated into a continuous amylose matrix. Amylose molecules that are dissolved during gelatinization reassociate to local double helices interconnected by hydrated parts of the molecules, and a continuous network (gel) forms [27]. As amylose retrogradation proceeds, double helix formation increases and, finally, very stable crystalline structures are formed, which cannot be melted again by heating. Amylopectin retrogradation takes several hours or days and occurs in the granule remnants embedded in the initial amylose gel [27]. Crystallization mainly occurs within the short-chain outer A- and B1-chains of the molecules. The amylopectin crystallites melt at ca. 60 °C and, therefore, aged bread can partly be “refreshed” by heating. This so-called “staling endotherm” can be measured by DSC to evaluate amylopectin retrogradation. Amylopectin retrogradation is strongly influenced by a number of conditions and substances, including pH and the presence of low-molecularweight (LMW) compounds such as salts, sugars, and lipids [26].

2.2.1.4

Interaction with Lipids

Amylose is able to form helical inclusion complexes in particular with polar lipids and this can occur in native (starch lipids; see below) as well as in gelatinized starch [28]. During gelatinization amylose forms a left-handed single helix and the nonpolar moiety of the polar lipid is located in the central cavity [16]. The inclusion complexes give rise to a V-type X-ray diffraction pattern. The presence of polar lipids strongly affects the retrogradation characteristics of the starch, because amylose-lipid complexes do not participate in the recrystallization process [26]. Complex formation is, however, strongly affected by the structure of the polar lipid [29]. For example, monoglycerides are more active than diglycerides and saturated fatty acids more active than unsaturated ones, because inclusion complexes are preferably formed with linear hydrocarbon chains and with compounds having one fatty acid residue. In addition, lipids, in particular lysophospholipids (lysolecithin), are minor constituents of cereal starches in amounts of 0.8–1.2% [30]. As so-called starch lipids they are associated with amylose as well as with the outer branches of amylopectin [28]. These lipid complexes lead to a delay of the onset of gelatinization and affect the properties of the starch especially in baking applications.

2.2.2

Nonstarch Polysaccharides (NSP)

Polysaccharides other than starch are primarily constituents of the cell walls and are much more abundant in the outer than in the inner layers of the grains. Therefore, a higher extraction rate is associated with a higher content of NSP. From a nutritional point of view NSP are dietary fiber, which has been associated with positive health effects. For example, cereal dietary fiber has been related to a reduced risk of chronic

2 Chemistry of Cereal Grains

17

life style diseases such as cardiovascular diseases, type II diabetes, and gastrointestinal cancer [31–36]. In addition, technological functionalities have been described for the arabinoxylans (AX) of wheat (reviewed by [4]) and rye.

2.2.2.1 Arabinoxylans AX are the major fraction (85–90%) of the so-called pentosans. Different cereal species contain different amounts of AX. The highest contents are present in rye (6–8%), whereas wheat contains only 1.5–2% AX. On the basis of solubility AX can be subdivided into a water-extractable (WEAX) and a water-unextractable fraction (WUAX). The former makes up 25–30% of total AX in wheat and 15–25% in rye [37]. In particular WEAX has considerable functionality in breadmaking. AX consist of linear b-(1,4)-d-xylopyranosyl-chains, which can be substituted at the O-2 and/or O-3-positions with a-l-arabinofuranose [38, 39]. A particular minor component of AX is ferulic acid, which is bound to arabinose as an ester at the O-5 position [40]. AX of different cereals may vary substantially in content, substitutional pattern and molecular weight [41–43]. WEAX mainly consist of two populations of alternating open and highly branched regions, which can be distinguished by their characteristic arabinose/xylose ratios, ranging between 0.3 and 1.1 depending on the specific structural region [44]. WUAX can be solubilized by mild alkaline treatment yielding structures that are comparable to those of WEAX [37, 45–48]. The unique technological properties of AX are attributable to the fact that AX are able to absorb 15–20 times more water than their own weight and, thus, form highly viscous solutions, which may increase gas holding capacity of wheat doughs via stabilization of the gas bubbles [49]. In total, WEAX bind up to 25% of the added water in wheat doughs [50]. Under oxidizing conditions, in particular under acidic pH, the so-called “oxidative gelation” [51] leads to AX gel formation by inducing di- and oligoferulic acid cross-links [52, 53]. This is thought to be one major structure-forming reaction in rye sourdoughs. Because of covalent crosslinks to the cell wall structure WUAX do not dissolve in water. Although they have high water-holding capacity and assist in water binding during dough mixing they are considered to have a negative impact on wheat breadmaking as they form physical barriers against the gluten network and, thus, destabilize the gas bubbles. However, the baking performance can be affected by adding endoxylanases, which preferentially hydrolyze WUAX. This produces solubilized WUAX, which have techno-functional effects comparable to WEAX [54, 55]. Beside AX the pentosan fraction contains a small part of a water-soluble, highly branched arabinogalactan peptide [41]. It consists of b-(1,3) and b-(1,6) linked galactopyranose units with a-glycosidically bound arabinofuranose residues. The peptide is attached by 4-trans-hydroxyproline. Unlike AX, arabinogalactan peptides have no significant effects in cereal processing.

P. Koehler and H. Wieser

18

2.2.2.2 b-Glucans b-Glucans are also called lichenins and are present particularly in barley (3–7%) and oats (3.5–5%), whereas less than 2% b-glucans are found in other cereals. The chemical structure of these NSP is made up of linear D-glucose chains linked via mixed b-(1,3)- and b-(1,4)-glycosidic linkages. b-Glucans show a higher water solubility than AX (38–69% in barley, 65–90% in oats) and form viscous solutions, which in the case of barley may interfere in wort filtration during the production of beer.

2.3

Proteins

The average protein content of cereal grains covers a relatively narrow range (8–11%, Table 2.2), variations, however, are quite noticeable. Wheat grains, for instance, may vary from less than 6% to more than 20%. The content depends on the genotype (cereal, species, variety) and the growing conditions (soil, climate, fertilization); amount and time of nitrogen fertilization are of particular importance. Proteins are distributed over the whole grain, their concentration within each compartment, however, is remarkably different. The germ and aleurone layer of wheat grains, for instance, contain more than 30% proteins, the starchy endosperm ~13%, and the bran ~7% [3]. Regarding the different proportions of these compartments, most proteins of grains are located in the starchy endosperm, which is the source of white flours obtained by milling the grains and sieving. White flours are the most important grain products. Therefore, the predominant part of the literature on cereal proteins deals with white flour proteins. The amino acid compositions of flour proteins from various cereals are shown in Table 2.3. Typical of all flours is the fact that glutamic acid almost entirely occurs in its amidated form as glutamine [56]. This amino acid generally predominates (15–31%), followed by proline in the case of wheat, rye, and barley (12–14%). Further major amino acids are leucine (7–14%) and alanine (4–11%). The nutritionally essential amino acids tryptophan (0.2–1.0%), methionine (1.3–2.9%), histidine (1.8–2.2%), and lysine (1.4–3.3%) are present only at very low levels. Through breeding and genetic engineering, attempts are being made to improve the content of essential amino acids. These approaches have been successful in the case of high-lysine barley and corn.

2.3.1

Osborne Fractions

Traditionally, cereal flour proteins have been classified into four fractions (albumins, globulins, prolamins, and glutelins) according to their different solubility and based on the fractionation procedure of Osborne [57]. Albumins are soluble in water,

19

2 Chemistry of Cereal Grains

Table 2.3 Amino acid composition (mol-%) of the total proteins of flours from various cereals [56] Amino acid Wheat Rye Barley Oats Rice Millet Corn Asxa Thr Ser Glxa Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp Amide group a

4.2 3.2 6.6 31.1 12.6 6.1 4.3 1.8 4.9 1.4 3.8 6.8 2.3 3.8 1.8 1.8 2.8 0.7 31.0

6.9 4.0 6.4 23.6 12.2 7.0 6.0 1.6 5.5 1.3 3.6 6.6 2.2 3.9 1.9 3.1 3.7 0.5 24.4

4.9 3.8 6.0 24.8 14.3 6.0 5.1 1.5 6.1 1.6 3.7 6.8 2.7 4.3 1.8 2.6 3.3 0.7 26.1

8.1 3.9 6.6 19.5 6.2 8.2 6.7 2.6 6.2 1.7 4.0 7.6 2.8 4.4 2.0 3.3 5.4 0.8 19.2

8.8 4.1 6.8 15.4 5.2 7.8 8.1 1.6 6.7 2.6 4.2 8.1 3.8 4.1 2.2 3.3 6.4 0.8 15.7

7.7 4.5 6.6 17.1 7.5 5.7 11.2 1.2 6.7 2.9 3.9 9.6 2.7 4.0 2.1 2.5 3.1 1.0 22.8

5.9 3.7 6.4 17.7 10.8 4.9 11.2 1.6 5.0 1.8 3.6 14.1 3.1 4.0 2.2 1.4 2.4 0.2 19.8

Asx Asp+Asn, Glx Glu+Gln

while globulins are insoluble in pure water but soluble in dilute salt solutions. Prolamins are classically defined as cereal proteins soluble in aqueous alcohols, for example 60–70% ethanol. Originally, glutelins were described as proteins that were insoluble in water, salt solution, aqueous alcohols and soluble in dilute acids or bases. Later, it was ascertained that notable portions of glutelins are insoluble in dilute acids such as acetic acid, and that extraction with strong bases destroys the primary structure of proteins. Nowadays, complete solubility of glutelins is achieved by solvents containing a mixture of aqueous alcohols (e.g., 50% propanol), reducing agents (e.g., dithiothreitol), and disaggregating compounds (e.g., urea). Regarding their functions, most of the albumins and globulins are metabolic proteins, for example enzymes or enzyme inhibitors (see Sect. 2.3.4). Oats are an exception containing considerable amounts of legume-like globulins such as 12S globulin [58]. Albumins and globulins are concentrated in the aleurone layer, bran, and germ, whereas their concentration in the starchy endosperm is relatively low. Predominantly, prolamins and glutelins are the storage proteins of cereal grains (see Sects. 2.3.2 and 2.3.3). Their only biological function is to supply the seedling with nitrogen and amino acids during germination. They are located only in the starchy endosperm; in white flours, their proportions based on total proteins amount to 70–90%. In general, none of the Osborne fractions consists of a single protein, but of a complex mixture of different proteins. A small portion of proteins does not fall into any of the four solubility fractions. Together with starch, they remain in the insoluble residue after Osborne fractionation and mainly belong to the class of lipo (membrane) proteins.

P. Koehler and H. Wieser

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The prolamin fractions of the different cereals have been given trivial names: gliadin (wheat), secalin (rye), hordein (barley), avenin (oats), zein (corn), kafirin (millet, sorghum), and oryzin (rice). The glutelin fraction of wheat has been termed glutenin. Terms for the other glutelin fractions such as secalinin (rye), hordenin (barley), and zeanin (corn) are scarcely used today. Gliadin and glutenin fractions of wheat have been combined in the terms gluten or gluten proteins. The content of the Osborne fractions varies considerably and depends on genotype and growing conditions. Moreover, the results of Osborne fractionation are strongly influenced by experimental conditions, and the fractions obtained are not clear-cut. Therefore, data from the literature on the qualitative and quantitative composition of Osborne fractions is differing and, in parts, contradictory. On average, the smallest proportion of total protein is present in the globulin fraction, followed by the albumin fraction. An exception is oat globulins amounting to more than 50% of total proteins. In most cereal flours, prolamins are the dominating fractions, oat prolamins, however, are minor protein components and rice flour is almost free of prolamins. Beside quantitative aspects the Osborne procedure is still useful for the preparation and characterization of flour proteins and the enrichment of different protein types.

2.3.2

Storage Proteins of Wheat Rye, Barley, and Oats

2.3.2.1

Classification and Primary Structures

Storage proteins (prolamins and glutelins) have been extensively investigated by the analysis of amino acid compositions, amino acid sequences, MW, and intra- and interchain disulfide linkages. The results indicated that, in accordance with phylogeny (see Sect. 2.1), the storage proteins of wheat, rye, and barley are closely related, whereas those of oats, in particular their glutelins, are structurally divergent. According to common structures storage proteins have been classified into three groups by two different principles. Shewry and coworkers [59] defined all storage proteins as prolamins and grouped them into the high-molecular-weight (HMW), sulfur-poor (S-poor) and sulfur-rich (S-rich) prolamins based on differences in MW and sulfur (cysteine, methionine) content. To prevent confusion, however, the term “prolamin” is not used for total storage proteins in the present paper, since classically the term prolamins comprises only the alcohol-soluble portions of storage proteins and does not include glutelins. We classified storage proteins according to related amino acid sequences and molecular masses into the following groups [60, 61]: (1) a HMW group; (2) a medium-molecular-weight (MMW) group; and (3) a LMW group. The proteins of these groups can be divided into different types on the basis of structural homologies (Table 2.4). Each type contains numerous closely related proteins; the small differences in their amino acid sequences can be traced back to substitutions, insertions, and deletions of single amino acids and short peptides.

21

2 Chemistry of Cereal Grains Table 2.4 Characterization of storage protein types from wheat, rye, barley, and oats [61, 63] Partial amino acid composition (mol-%)b Repetitive unitb,c Group/type HMW group HMW-GS x HMW-GS y HMW-secalin x HMW-secalin y D-hordein MMW group w5-gliadin w1,2-gliadin w-secalin C-hordein LMW group a/ß-gliadin

Code

Residues Statea (frequency)

Q P

Q6R2V1 Q52JL3 Q94IK6 Q94IL4 Q40054

815 637 760 716 686

a a a a a

QQPGQG (72×) QQPGQG (50×) QQPGQG (66×) QQPGQG (60×) QQPGQG (26×)

36 32 34 34 26

13 11 15 12 11

Q402I5 Q6DLC7 O04365 Q40055

420 373 338 328

m m m m

(Q)QQQFP (65×) (QP)QQPFP (42×) (Q)QPQQPFP (32×) (Q)QPQQPFP (36×)

53 42 40 37

20 10.0 0.7 3.1 0.2 29 9.9 0.8 4.0 0.5 29 8.6 0.6 4.4 1.8 29 9.4 0.6 8.6 0.3

Q9M4M5 273

m

QPQPFPPQQPYP (5×) (Q)QPQQPFP (15×) (Q)QQPPFS (11×) QPQQPFP QQPQQPFP (32×) QPQQPFP (15×) QQPFPQ (13×) PFVQQQQ (3×)

36 15

F +Y G

g-gliadin Q94G91 308 m 36 18 LMW-GS Q52NZ4 282 a 32 13 g-40 k-secalind – – m 34 18 g-75 k-secalin Q9FR41 436 a 38 22 g-hordein P17990 286 m 28 17 B-hordein P06470 274 a 30 19 avenin Q09072 203 m 33 11 a a aggregative, m monomeric b One-letter-code for amino acids c Basic unit frequently modified by substitution, insertion, and deletion of residues d Gellrich et al. [65]

5.8 5.5 6.7 5.0 5.5

20 18 20 18 16

L

V

4.4 3.8 3.7 3.2 4.1

1.7 2.3 1.5 1.8 4.1

7.4 2.6 8.1 5.1 5.2 5.7 5.5 6.1 7.7 7.3 8.4

2.9 3.2 2.4 1.6 3.1 2.9 2.0

7.2 8.2 7.4 4.8 7.0 8.0 8.9

4.6 5.3 4.7 5.3 7.3 6.2 8.3

single amino acid

The nomenclature of types is rather confusing and inconsequential. On the one hand prolamins have been termed according to their electrophoretic mobility in acid polyacrylamide gel electrophoresis (PAGE) with band regions designated as w (lowest mobility), g (medium mobility), and a/b (highest mobility). On the other hand, the nomenclature is based on their apparent sizes (after reduction of disulfide bonds) as indicated by sodiumdodecyl sulfate (SDS-) PAGE; examples are HMWand LMW-glutenin subunits (GS), HMW-secalins, D-, C-, and B-hordeins. Because of the different importance of HMW-GS for the bread-making quality of wheat, single subunits have been numbered according to their mobility on SDS-gel electrophoresis (original nos. 1–12), the genome (1A, 1B, or 1D), and the type (x or y); examples of nomenclature are HMW-GS 1Ax1, 1Bx7, and 1Dy10 [62]. The HMW group contains HMW-GS of wheat, HMW-secalins of rye, and D-hordeins of barley (Table 2.4); this type is missing in oats. HMW-GS and HMWsecalins can be subdivided into the x-type and the y-type. The proteins comprise around 600–800 amino acid residues corresponding to MW of 70,000–90,000.

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Fig. 2.1 Schematic structure and disulfide bonds of a/b-gliadins, g-gliadins, LMW-, and HMW-GS (Adapted from [64])

The amino acid compositions are characterized by high contents of glutamine, glycine, and proline. The amino acid sequences [63] can be separated into three structural domains: a nonrepetitive N-terminal domain A of ~100 residues, a repetitive central domain B of 400–700 residues, and a nonrepetitive C-terminal domain C with ~40 residues (Fig. 2.1) [64, 65]. Domain B is dominated by repetitive sequences such as QQPGQG (one-letter-code for amino acids) as a backbone with inserted sequences like YYPTSL, QQG, and QPG with remarkable differences between x- and y-types (Table 2.5). Domains A and C have a more balanced amino acid composition and more amino acid residues with charged side chains. In a native state, the proteins of the HMW group are aggregated through interchain disulfide bonds (Fig. 2.1). The MMW group consists of the homologous w1,2-gliadins of wheat, w-secalins of rye, and C-hordeins of barley including amino acid residues between 300 and 400 and MW around 40,000 (Table 2.4). Additionally, wheat contains unique w5gliadins with more than 400 residues and MW around 50,000. This group, likewise, is not present in oats. The proteins of the MMW group have extremely unbalanced amino acid compositions characterized by high contents of glutamine, proline, and phenylalanine, which together account for ~80% of total residues. Most sections of the amino acid sequences are composed of repetitive units such as QPQQPFP or QQQFP. This type of protein occurs as monomers and is readily soluble in aqueous alcohols and, in parts, even in water.

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Table 2.5 Partial amino acid sequences of domain B of HMW-GS 1Dx2 (positions 93–338) and of HMW-GS 1Dy10 (positions 106–380) [63] Position Sequencea Position Sequencea 93 YYPSVTSPQQVS 106 YYPGVTSPRQGS 105 YYPGQASPQRPGQG 118 YYPGQASPQQPGQG 119 QQPGQG 132 QQPGKW 125 QQSGQGQQG 138 QEPGQGQQW 134 YYP--TSPQQPGQW 147 YYP--TSLQQPGQG 146 QQPEQGQPG 159 QQIGKGQQG 155 YYP--TSPQQPGQL 168 YYP--TSLQQPGQGQQG 167 QQPAQG 183 YYP--TSLQHTGQR 173 QQPGQGQQG 195 QQPVQG 182 QQPGQGQPG 201 QQPEQG 191 YYP-TSSQLQPGQL 207 QQPGQWQQG 204 QQPAQGQQG 216 YYP--TSPQQLGQG 213 QQPGQGQQG 228 QQPRQW 222 QQPGQG 234 QQSGQGQQG 228 QQPGQGQQG 243 HYP--TSLQQPGQGQQG 237 QQPGQG 258 HYL--ASQQQPGQGQQG 243 QQPGQGQQG 273 HYP--ASQQQPGQGQQG 252 QQLGQGQQG 288 HYP--ASQQQPGQGQQG 261 YYP--TSLQQSGQGQPG 303 HYP--ASQQEPGQGQQG 276 YYP--TSLQQLGQGQSG 318 QIPASQ 291 YYP--TSPQQPGQG 324 QQPGQGQQG 303 QQPGQL 333 HYP--ASLQQPGQGQQG 309 QQPAQG 348 HYP--TSLQQLGQGQQT 315 QQPGQGQQG 363 QQPGQK 324 QQPGQGQQG 369 QQPGQG 333 QQPGQG 375 QQTGQG a One-letter-code for amino acids; - deletion

The LMW group consists of monomeric proteins including a/b- and g-gliadins of wheat, g-40 k-secalins of rye, g-hordeins of barley, and avenins of oats, and of aggregative proteins including LMW-GS of wheat, g-75 k-secalins of rye, and B-hordeins of barley (Table 2.4). They have around 300 amino acid residues and MW ranging from 28,000–35,000, besides g-75 k-secalins (~430 residues, MW ~50,000) and avenins (~200 residues, MW ~23,000). The amino acid compositions of the LMW group proteins are characterized by relatively high contents of hydrophobic amino acids besides glutamine and proline. The amino acid sequences consist of four (a/b-gliadins five) different sequence sections (Fig. 2.1). The N-terminal section I is rich in glutamine, proline, and phenylalanine forming repetitive units such as QPQPFPPQQPY (a/b-gliadins), QQPQQPFP (g-gliadins), QQPPFS (LMW-GS), or PFVQQQQ (avenins). Section I of g-75 k-secalins is prolonged by around 130 residues as compared to g-40 k-secalins and that of avenins is shortened to around 40 residues. Section II is unique to a/b-gliadins and consists of a polyglutamine sequence (up to 18 Q-residues). Sections III, IV, and V possess more balanced

P. Koehler and H. Wieser

24

amino acid compositions and most of the cysteine residues that form only intrachain disulfide bonds (monomeric proteins) or both intra- and interchain disulfide bonds (aggregative proteins). The comparison of the amino acid sequences demonstrates that sections III and V contain homologous sequences, whereas section IV is, in part, unique to each type (Table 2.6). g-Type proteins (g-gliadins, g-40 k-secalins [66], g-75 k-secalins, g-hordeins) show the highest conformity; a/b-gliadins, LMW-GS, and avenins have the lowest degree of homology within the LMW group. Most oat glutelins are globulin-like proteins and do not show any structural relationship with the HMW-, MMW-, and LMW-type proteins described above [67]. The reasons as to why they are not extractable with a salt solution are not yet clear. As mentioned earlier, the quantitative composition of storage protein is strongly dependent on both genotype and growing conditions. Nevertheless, some constant data can be observed (Table 2.7) [68–70]: Proteins of the LMW group belong, by far, to the major components. Within this group, monomeric proteins (55–77% of total storage proteins) exceed aggregative proteins (10–25%) in the case of wheat species, whereas rye and barley are characterized by more aggregative (34–48%) than monomeric proteins (~25%). Proteins of the MMW and HMW groups belong to the minor components except w-secalins (18%) and C-hordeins (36%) or are missing (oats). Within wheat species significant differences can be observed. Common wheat is characterized by the highest values for aggregative proteins (HMW-, LMW-GS) and a low monomeric/aggregated (m/a) ratio, and the “old” wheat species emmer and einkorn by low proportions of HMW-GS and high m/a ratios.

2.3.2.2

Disulfide Bonds

Disulfide bonds play an important role in determining the structure and properties of storage proteins. They are formed between sulfhydryl groups of cysteine residues, either within a single protein (intrachain) or between proteins (interchain). Most information on disulfide bonds is available for wheat gliadins and glutenins. With a few exceptions, w-type gliadins are free of cysteine and, consequently occur as monomers. Most a/b- and g-gliadins contain six and eight cysteine residues, respectively, and form three or four homologous intrachain disulfide bonds, present within or between sections III and V (Fig. 2.1) [64]. A small portion of gliadins is different from most gliadins and contains an odd number of cysteine residues. They may be linked to each other or to glutenins by an interchain disulfide bond. Homologous to g-gliadins, g-40 k- and g-75 k-secalins, g- and B-hordeins as well as avenins contain eight cysteine residues at comparable positions within sections III–V (Table 2.6). Probably they form four intrachain disulfide bonds homologous to those of g-gliadins (Fig. 2.2). LMW-GS include eight cysteine residues, six of which form three intrachain disulfide bonds homologous to those of a/b- and g-gliadins [64, 65]. Two cysteine residues located in sections I and IV are unique to LMW-GS; they are obviously not able to form intrachain bonds for steric reasons. They are involved in interchain bonds with residues of different proteins (LMW-GS, modified gliadins, y-type HMW-GS).

Table 2.6 Amino acid sequences of sections III, IV, and V of the LMW group [63] Type Positions Sequences (a) Section III a/b-gliadin 119–186 ILQQILQQQLIPCMDVVLQQHNIVHGRSQVLQQSTY-----QLLQELCCQHLWQIPEQSQCQAIHNVVHAIIL g-gliadin 153–224 FIQPSLQQQLNPCKNILLQQCKPASLVSSL-WSIIWPQSDCQVMRQQCCQQLAQIPQQLQCAAIHSVVHSIIM LMW-GS 101–173 IVQPSVLQQLNPCKVFLQQQCSPVAMPQRLARSQMWQQSRCHVMQQQCCQQLSQIPEQSRYDAIRAITYSIIL g-40 k-secalina – SIQLSLQQQLNPCKNVLLQQCSPVALVSSL-RSKIFPQSECQVMQQQCCQQLAQIPHHLQCAAIHSVVHAIIM g-75 k-secalin 285–356 SIQLSLQQQLNPCKNVLLQQCSPVALVSSL-RSKIFPQSECQVMQQQCCQQLAQIPQQLQCAAIHSVVHAIIM g-hordein 135–206 TIQLYLQQQLNPCKEFLLQQCRPVSLLSYI-WSKIVQQSSCRVMQQQCCLQLAQIPEQYKCTAIDSIVHAIFM B-hordein 112–184 YVHPSILQQLNPCKVFLQQQCSPVPVPQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQFRHEAIRAIVYSIFL Avenin 43–114 FLQPLLQQQLNPCKQFLVQQCSPVAAVPFL-RSQILRQAICQVTRQQCCRQLAQIPEQLRCPAIHSVVQSIIL (b) Section IV a/b-gliadin 187–222 HQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQN g-gliadin 225–253 QQQQQQQQQQGMHIFLPLSQQQQVGQGSL LMW-GS 174–228 QEQQQGFVQAQQQQPQQSGQGVSQSQQQSQQQLGQCSFQQPQQQLGQQPQQQQQQ g-40 k-secalina – QQEQREGVQILLPQSHQQLVGQGAL g-75 k-secalin 357–381 QQEQREGVQILLPQSHQQHVGQGAL g-hordein 207–231 QQGQRQGVQIVQQQPQPQQVGQCVL B-hordein 185–225 QEQPQQLVEGVSQPQQQLWPQQVGQCSFQQPQPQQVGQQQQ Avenin 115–155 QQQQQQQQFIQPQLQQQVFQPQLQLQQQVFQPQLQQQVFQP (c) Section V a/b-gliadin 223–273 PQAQGSVQPQQLPQF-EEIRNLALQTLPAMCNVYIPPYCTI--APFGIFGTNYR g-gliadin 254–308 VQGQGIIQPQQPAQL-EAIRSLVLQTLPSMCNVYVPPECSIMRAPFASIVAGIGGQ LMW-GS 229–282 VLQGTFLQPHQIAHL-EAVTSIALRTLPTMCSVNVPLYSATTSVPFAVGTGVSAY g-40 k-secalina – AQVQGIIQPQQLSQFNVGIVLQMLQNLPTMCNVYVPRQCPPSRRHLHAMSLVCGH g-75 k-secalin 382–436 AQVQGIIQPQQLSQL-EVVRSLVLQNLPTMCNVYVPRQCSTIQAPFASIVTGIVGH g-hordein 232–286 VQGQGVVQPQQLAQM-EAIRTLVLQSVPSMCNFNVPPNCSTIKAPFVGVVTGVGGQ B-hordein 226–274 VPQSAFLQPHQIAQL-EATTSIALRTLPMMCSVNVPLYRILRGVGPSVGV Avenin 156–203 QLQQVFNQPQMQGQI-EGMRAFALQALPAMCDVYVPPQCPVATAPLGGF a Gellrich et al. [65]

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Table 2.7 Proportions (%) of storage protein types of different wheat species, rye, and barley [68–70] Group HMW MMW LMW Cereal Variety a m a m m/aa Common wheat Rektor 9.1 10.4 25.1 55.4 1.9 Spelt wheat Schwabenkorn 6.6 10.4 17.7 65.3 3.1 Durum wheat Biodur 5.0 6.7 19.3 69.0 3.1 Emmer Unknown 2.6 10.8 10.0 76.6 6.9 Einkorn Unknown 3.5 12.8 19.3 64.5 3.4 Rye Halo 9.0 17.6 48.4 25.0 0.7 Barley Golden promise 5.0 35.8 34.1 25.1 1.6 a a aggregative, m monomeric

Fig. 2.2 Schematic two-dimensional structures of the C-terminal domain (sections III–V) of g-gliadins (Taken from [94])

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Because HMW-GS do not occur as monomers, it is generally assumed that they form interchain disulfide bonds. The x-type subunits, except subunit 1Dx5, have three cysteine residues in domain A and one in domain C (Fig. 2.1). Cysteines Ca and Cb were found to be linked by an intrachain bond, thus, the others (Cd, Cz) are available for interchain bonds. Subunit 1Dx5 has an additional cysteine residue at the beginning of domain B, and it has been suggested that this might form another interchain bond. Recently, a so-called head-to-tail disulfide bond between HMW-GS has been identified [71]. The y-type subunits have five cysteine residues in domain A and one in each of domains B and C. At present, interchain linkages have only been found for adjacent cysteine residues of domain A (Cc1, Cc2), which are connected in parallel with the corresponding residues of another y-type subunit, and for cysteine Cy of domain B, which is linked to Cx of section IV of LMW-GS. Thus, HMW- and LMW-GS fulfill the requirement that at least two cysteines forming interchain disulfide bonds are necessary to participate in a growing polymer; they act as “chain extenders.” The most recent glutenin model suggests a backbone formed by HMW-GS linked by end-to-end, probably head-to-tail interchain disulfide bonds [65]. LMW-GS form also linear polymers via cysteine residues of sections I and IV; they are linked to domain B of y-type HMW-GS. y-Type HMW-secalins of rye have a second cysteine in domain C, which opens the possibility that an intrachain disulfide bond within domain C is formed inhibiting an interchain bond for polymerization [72]. As far as is known, D-hordeins possess ten (!) cysteine residues [63]; the formation of a regular polymer backbone appears to be impossible.

2.3.2.3

Molecular Weight Distribution

Most information on the quantitative MW distribution (MWD) of native storage (gluten) proteins is available for wheat, because MWD of gluten proteins has been recognized as one of the main determinants of the rheological properties of wheat dough. Native gluten proteins consist of monomeric a/b- and g-gliadins with MW around 30,000 and monomeric w5- and w1,2-gliadins with MW between 40,000 and 55,000. They are alcohol-soluble and amount to ~50% of gluten proteins (Fig. 2.3). Besides monomers the alcohol-soluble fraction contains oligomers with MW roughly ranging between 60,000 and 600,000. They are formed by modified gliadins with an odd number of cysteine residues and LMW-GS via interchain disulfide bonds and account for ~15% of gluten proteins. Composition and quantity of the oligomeric fraction are strongly determined by the conditions of alcohol extraction, for example by temperature and duration. The remaining proteins (~35%) are alcohol-insoluble and mainly composed of LMW-GS and HMW-GS linked by disulfide bonds. Their MW ranges approximately from 600,000 to more than 10 million. The largest polymers termed “glutenin macropolymers” (GMP) are insoluble in SDS solutions and have MW well into the multimillions indicating that they may belong to the largest proteins in nature [73, 74]. Their amounts in flour (20–40 mg/g) are strongly correlated with dough strength and bread volume. GMP is characterized by higher ratios of HMW-GS to LMW-GS and x-type to y-type HMW-GS in

P. Koehler and H. Wieser

28 Fig. 2.3 Molecular weight distribution of native wheat storage (gluten) proteins (Modified from [73])

comparison with total glutenins; the HMW-GS combination 1Dx5 + 1Dy10 appears to produce higher GMP concentrations than 1Dx2 + 1Dy12 [73]. Rye storage proteins have a strongly different MWD as compared to wheat. Although rye shows higher proportions of aggregative to monomeric storage proteins than wheat (Table 2.7), the proportions of polymers is much lower (~23%) and the amount of GMP (~5 mg/g flour) strongly reduced [75, 76]. The deficiency of polymeric proteins is balanced by the higher proportion of oligomers (~30%), whereas the proportion of rye monomers (~47%) is similar to that of wheat. Obviously rye storage proteins consist of many more chain terminations (e.g., g-75 k-secalins, y-type HMW-secalins) and less chain extenders than wheat, which apparently prevents gluten formation during dough mixing. Information about the MWD of native barley and oat proteins is not yet available. 2.3.2.4

Influence of External Parameters

Many studies have substantiated that both structures and quantities of storage proteins are exposed to a continuous change from the growing period of plants to the processing of end products. Because of the importance of wheat as a unique “bread cereal,” most investigations have been focused on gluten proteins. In principle, however, the effect of external parameters is similar for all cereal proteins. Fertilization The supply with minerals during growing is essential for optimal plant development. Nitrogen (N) fertilization is, in particular, important for common wheat, because a high N supply provides a high flour protein content and thus, increased bread volume. Fertilization with different N amounts demonstrated that the quantities

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of albumins/globulins are scarcely influenced, whereas those of gluten proteins increase with higher N supply [77]. The effects on gliadins are more pronounced than on glutenins resulting in an elevated gliadin/glutenin ratio. Particularly, the proportions of w-type gliadins are strongly enhanced by high N supply. In the past, sulfur (S)-containing fertilizers were not widely used for cereal crops, because air pollution from industry and traffic provided sufficient amounts of S in the soil. The massive decrease in the input of S from atmospheric deposition over the last decades reduced S availability in soils dramatically, and has led to a severe S deficiency in cereals, which exerts a large influence on protein composition and technological properties. In the case of wheat, S deficiency provokes a drastic increase of S-free w-type gliadins and a decrease of S-rich g-gliadins and LMW-GS [78]. Moreover, S deficiency has been reported to impair dough and bread properties [79]. Infections The infection of cereal plants with Fusarium strains induces a premature fading of individual spikelets and then, a fading of the whole ears. An outbreak of this infection is often accompanied by mycotoxin contamination of grains and flours, for example by the trichothecene deoxinivalenol. Studies on wheat demonstrated that infection with Fusarium caused a distinct reduction in the content of both total glutenins and HMW-GS and impaired dough and bread quality [80]. Glutenins of common wheat have been shown to be more strongly affected than those of emmer and storage proteins of naked barley [81, 82].

Germination Proteins as well as other constituents are stable in dry grains. Water supply, however, induces germination of grains accompanied by the activation of enzymes, in particular, amylases and peptidases. The latter have been shown, for instance, to cause a fast degradation of prolamins of wheat, rye, barley, and oats during germination [83]. Studies on the Osborne fractions of wheat demonstrated that both monomeric gliadins and polymeric glutenins were strongly degraded during germination for 168 h at 15–30 °C, whereas albumins/globulins were scarcely affected [84]. The degradation of gluten proteins has a drastic negative effect on the bread-making quality of wheat.

Oxidation Grains contain a considerable amount of LMW thiols such as glutathione, which are known to affect the structures and functional properties of polymeric storage proteins by thiol/disulfide interchange reactions during dough preparation [64]. To prevent this deleterious effect on the bread-making quality of wheat, week-long storage of

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flour under air (direct oxidation of LMW thiols) and treatment with L-ascorbic acid (indirect oxidation catalyzed by glutathione dehydrogenase) are recommended.

Enzymes Breads prepared from rye and wheat sourdoughs are of increasing consumer interest due to the improvement of sensorial and nutritional quality, the prolongation of shelf life, and the delay in staling. Wheat storage proteins, however, which are responsible for the viscoelastic and gas-holding properties of dough and for the texture of the bread crumb, are profoundly degraded [85]. Protein degradation during fermentation is primarily due to acidic peptidases present in flour and activated by the lowered pH caused by Lactobacillus strains. The strongest decrease was found for the glutenin macropolymer and total glutenins. The extent of the decrease of monomeric gliadins was lower and more pronounced for the g-type than for the a/b- and w-types.

Heat The baking process involves a drastic heat-treatment of proteins, with temperatures of more than 200 °C on the outer layer (crust) and near 100 °C in the interior (crumb) of bread. HPLC analysis of crust proteins from wheat bread indicated serious structural damage of both gliadins and glutenins [86]. With respect to crumb, the extractability of total gliadins with 60% ethanol is strongly reduced compared with those from flours. The single gliadin types are affected differently, w-type gliadins less and a/b- and g-types much more. Most gliadins can be recovered in the glutenin fraction after reduction of disulfide bonds suggesting that major heat-induced crosslinks of gliadins to glutenins are disulfide bonds.

High Pressure The effect of hydrostatic pressure is similar to that of heat. Treatment of gluten with pressure in the range of 300–600 MPa at 60 °C for 10 min provokes a strong reduction of gliadin extractability [87]. Within gliadin types, cysteine containing a/b- and g-type gliadins, but not cysteine-free w-type gliadins, are sensitive to pressure and are transferred to the ethanol-insoluble glutenin fraction. Cleavage and rearrangement of disulfide bonds have been proposed as being responsible for pressureinduced aggregation.

2.3.2.5 Wheat Gluten Wheat is unique among cereals in its ability to form a cohesive, viscoelastic dough, when flour is mixed with water. Wheat dough retains the gas produced during

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fermentation and this results in a leavened loaf of bread after baking. It is commonly accepted that gluten proteins (gliadins and glutenins) decisively account for the physical properties of wheat dough. Both protein fractions are important contributors to these properties, but their functions are divergent. Hydrated monomeric and oligomeric proteins of the gliadin fraction have little elasticity and are less cohesive than glutenins; they contribute mainly to the viscosity and extensibility of dough. In contrast, hydrated polymeric glutenins are both cohesive and elastic, and are responsible for dough strength and elasticity. Thus, gluten is a “two-component glue,” in which gliadins can be understood as a “plasticizer” or “solvent” for glutenins [65]. A proper mixture (~2:1) of the two is essential to give desirable dough and bread properties. Native gluten proteins are amongst the most complex protein networks in nature due to the presence of several hundred different protein components. Even small differences in the qualitative and quantitative protein composition decide on the end-use quality of wheat varieties. Numerous studies demonstrated that the total amounts of gluten proteins (highly correlated with the protein content of flour), the ratio of gliadins to glutenins, the ratio of HMW-GS to LMW-GS, the amount of GMP, and the presence of specific HMG-GS determine dough and bread quality. Amongst chemical bonds disulfide linkages (Fig. 2.1) play a key role in determining the structure and properties of gluten proteins. Intrachain bonds stabilize the steric structure of both monomeric and aggregative proteins; interchain bonds provoke the formation of large glutenin polymers. The disulfide structure is not in a stable state, but undergoes a continuous change from the maturing grain to the end product (e.g., bread), and is chiefly influenced by redox reactions. These include (1) the oxidation of free SH groups to S-S linkages, which supports the formation of large aggregates, (2) the presence of chain terminators (e.g., glutathione and gliadins with an odd number of cysteine), which stop polymerization, and (3) SH-SS interchange reactions, which affect the degree of polymerization of glutenins. Consequently, oxygen is known to be essential for optimal dough development and oxidizing agents, for example potassium bromide, azodicarbonimide, and dehydroascorbic acid (the oxidation product of ascorbic acid) have been found to be useful as bread improvers [88]. Conversely, reducing agents such as cysteine and sodium metabisulfite are used to soften strong doughs, accompanied by decreased dough development and resistance and increased extensibility. They are specifically in use as dough softeners for biscuits. The overall effect is to reduce the average MW of glutenin aggregates by SH/SS interchange. Beside disulfide bonds, dityrosine and isopeptide bonds have been described as further covalent cross-links between gluten proteins. Compared with the concentration of disulfide bonds (~10 mmol per g flour) tyrosine-tyrosine cross-links (~0.7 nmol per g flour) appear to be only of marginal importance [89]. Interchain cross-links between lysine and glutamine residues (isopeptide bonds) are catalyzed by the enzyme transglutaminase (TG). Addition of TG to flour results in a decrease in the quantity of extractable gliadins and an increase of the glutenin fraction and the nonextractable fraction [90]. Thereby, dough properties and bread-making quality can be positively influenced, similar to the actions of chemical oxidants.

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Fig. 2.4 A model double unit for the interchain disulfide structure of LMW-GS and HMW-GS of glutenin polymers (Adapted from [65])

The covalent structure of gluten proteins is complemented by noncovalent bonds (hydrogen bonds, ionic bonds, hydrophobic bonds). Glutamine, predestinated for hydrogen bonds, is the most abundant amino acid in gluten proteins (Table 2.4) and chiefly responsible for the water-binding capacity of gluten. In fact, dry gluten absorbs about twice its own weight of water. Moreover, glutamine residues are involved in frequent protein-protein hydrogen bonds. Though the number of ionizable side chains is relatively low, ionic bonds are of importance for the interactions between gluten proteins. For example, salts such as NaCl are known to strengthen dough, obviously via ionic bonds with glutenins [91]. Hydrophobic bonds can also contribute to the properties of gluten. Because the energy of hydrophobic bonds increase with increased temperature, this type of noncovalent bonds is particularly important for protein interactions during the oven phase. Both covalent and noncovalent bonds determine the native steric structures (conformation) of gliadins and glutenins. Studies on the secondary structure have indicated that the repetitive sequences of gliadins and LMW-GS are characterized by b-turn conformation, whereas the nonrepetitive sections contain considerable proportions of a-helix and b-sheet structures [92]. The nonrepetitive sections of a/b-, g-gliadins, and LMW-GS include intrachain disulfide bonds, which are concentrated in a relatively small area and form compact structures including two or three small rings and a big ring (Fig. 2.2) [93]. The nonrepetitive sections A and C of HMW-GS are dominated by a-helix and b-sheet structures, whereas the repetitive section B is characterized by regularly repeated b-turns [94]. They form a loose b-spiral similar to that of mammalian connective tissue elastin; b-spirals have been proposed to transfer elasticity to gluten.

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A range of models has been developed to explain the structure and functionality of glutenins. Most recently, the experimental findings on disulfide bonds were transformed into a two-dimensional model [65] (Fig. 2.4). HMW-GS and LMW-GS polymerize separately, both forming linear backbone polymers. Both polymers are cross-linked by a disulfide bond between section IV of LMW-GS and section B of y-type HMW-GS. The backbone of HMW-GS is established by end-to-end, probably head-to-tail linkages. LMW-GS polymers are linked between two sections I and between sections I and IV. The polymerization of HMW-GS and LMW-GS is terminated by chain terminators, either by modified gliadins or LMW thiol compounds.

2.3.3

Storage Proteins of Corn, Millet, Sorghum, and Rice

Overall, the storage proteins of corn, sorghum, millet, and rice are, in part, related and differ significantly from those of wheat, rye, barley, and oats. According to the amino acid composition they contain less glutamine and proline and more hydrophobic amino acids such as leucine [56]. Corn storage proteins, called zeins, can be subgrouped into alcohol-soluble monomeric zeins and cross-linked zeins alcoholsoluble only on heating or after reduction of disulfide bonds. With respect to different structures zeins have been divided into four different subclasses [95]. a-Zeins are the major subclass (71–85% of total zeins), followed by g- (10–20%), b- (1–5%) and d-zeins (1–5%), respectively [96]. a-Zeins are monomeric proteins with apparent MW of 19,000 and 22,000 determined by SDS-PAGE. Their amino acid sequences contain up to ten tandem repeats [97]. Proteins of the other subclasses are cross-linked by disulfide bonds and their subunits have apparent MW of 18,000 and 27,000 (g-zein), 18,000 (b-zein), and 10,000 (d-zein). In many ways the storage proteins of sorghum and millet called kafirins are similar to zeins. Sorghum kafirins have also been subdivided into a, b-, g- and d-subclasses based on solubility, MW, and structure [98]. a-Kafirins are monomeric proteins and represent the major subclass accounting for around 65–85% of total kafirins. Proteins of the other subclasses are highly cross-linked and alcohol-soluble only after reduction of disulfide bonds. On average, each of them accounts for less than 10% of total kafirins [99, 100]. Within the numerous millet species and varieties the proteins of foxtail millet were studied in detail [101]. SDS-PAGE of unreduced kafirins revealed bonds with apparent MW ranging from 11,000 to 150,000. After the reduction of disulfide bonds two major bands with MW of 11,000 (subunit A) and 16,000 (subunit B) were obtained. Unreduced proteins with higher MW were formed by cross-links of A and/or B subunits. The storage proteins of rice are characterized by the highly unbalanced ratio of prolamins to glutelins (~1:30) [102]. Both fractions show the lowest proline content (~5 mol-%) amongst cereal storage proteins [56]. SDS-PAGE patterns of rice prolamins (oryzins) showed a major band with MW 17,000 and a minor band with MW 23,000 [103]. The apparent MW of glutelin subunits was in a range from 20,000 to 38,000.

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2.3.4

Metabolic Proteins

Most proteins of the albumin and globulin fractions are metabolic proteins, mainly enzymes and enzyme inhibitors. The corresponding extensive studies have been summarized by Kruger and Reed [104] and recently by Delcour and Hoseney [105]. Many of these proteins are located in the embryo and aleurone layer; others are distributed throughout the endosperm. They have nutritionally better amino acid compositions than storage proteins, particularly because of their higher lysine contents. Those enzymes that hydrolyze carbohydrates and proteins and, thereby, provide the embryo with nutrients and energy during germination, are of most significant importance.

2.3.4.1

Hydrolyzing Enzymes

Carbohydrate-Degrading Enzymes The many carbohydrate-degrading enzymes include a-amylases, b-amylases, debranching enzymes, cellulases, b-glucanases, and glucosidases. Amylases are enzymes that hydrolyze the polysaccharides in starch granules. They can be classified as endohydrolases, which attack glucosidic bonds within the polysaccharide molecules and exohydrolases, which attack glucosidic bonds at or near the end of chains. The most important enzyme of the endohydrolase type is a-amylase. The enzyme hydrolyzes a-1,4-glucosidic bonds of amylose and amylopectin and produces a mixture of dextrins together with smaller amounts of maltose and oligosaccharides; the pH-optimum is about 5. The other major amylase type is b-amylase, an exohydrolase, which hydrolyzes a-1,4-glucosidic bonds near the nonreducing ends of amylose and amylopectin to produce maltose. Its pHoptimum is similar to that of a-amylase. Both amylase types exist in multiple forms or isoenzymes with different chemical and physical properties. Neither a- nor b-amylase can break a-1,6-glucosidic bonds present in amylopectin. For this kind of hydrolysis debranching enzymes are present in cereal grains. Along with a-glucosidases they assist a- and b-amylases in a more complete conversion of starch to simple sugars and small dextrins. A number of other carbohydrate degrading enzymes exist, their amounts, however, are very low compared to amylases. Examples are a- and b-glucosidases, cellulases, and arabinoxylanases.

Proteolytic Enzymes Enzymes that hydrolyze proteins are called proteinases, proteases, or peptidases. They attack the peptide bond between amino acid residues and include both endoand exopeptidases. The latter are divided into carboxypeptidases, when acting from the carboxy terminal and aminopeptidases, when acting from the amino terminal.

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The most important proteolytic enzymes are acidic peptidases. They exist in multiple forms having pH-optima between 4.2 and 5.5 and include both endo- and exotypes. On the basis of their catalytic mechanism they can be classified as serine, metallo-, aspartic, and serine peptidases. According to their biological function to provide the embryo with amino acids, their activity is highest during the germination of grains.

Other Hydrolyzing Enzymes Lipases are the most important enzymes that hydrolyze ester bonds. They attack triacylglycerols yielding mono- and diacylglycerols and free fatty acids. Lipase activity is important, because free fatty acids are more susceptible to oxidative rancidity than fatty acids bound in triacylglycerols. The activity varies widely among cereals with oats and millet having the highest activity. Exogenous lipases are in use to improve the baking performance of wheat flour. Phytase is an esterase that hydrolyzes phytic acid to inositol and free phosphoric acid. Even partial hydrolysis of phytic acid by phytase is desirable from a nutritional point of view, because the strong complexation of cations such as zinc, calcium, and magnesium ions by phytic acid is significantly reduced.

2.3.4.2

Oxidizing Enzymes

Lipoxygenase is present in high levels in the germ. It catalyzes the peroxidation of certain polyunsaturated fatty acids by molecular oxygen. Its typical substrate is linoleic acid containing a methylene-interrupted, doubly unsaturated carbon chain with double bonds in the cis-configuration. Polyphenoloxidases preferably occur in the outer layers of the grains. They catalyze the oxidation of phenols, such as catechol, pyrogallol, and gallic acid, to quinons by molecular oxygen. Peroxidase and catalase may be classified as hydroperoxidases catalyzing the oxidation of a number of aromatic amines and phenols, for example ferulic acid in arabinoxylans, by hydrogen peroxide. Other oxidizing enzymes are ascorbic acid oxidase and glutathione dehydrogenase.

2.3.4.3

Enzyme Inhibitors

Many investigators have isolated and characterized enzyme inhibitors from germ and endosperm. Most important inhibitors are targeted on hydrolyzing enzymes to prevent the extensive degradation of starch and storage proteins during grain development and to defend plant tissues from animal (insect) or microbial enzymes. Predominant classes are amylase and protease inhibitors concentrated in the albumin/globulin fractions. Amylase inhibitors can be directed towards both cereal and noncereal amylases and protease inhibitors towards proteases from both cereals and

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animals. Some inhibitors appear to be bifunctional inhibiting amylases as well as proteases.

2.4 2.4.1

Lipids Lipid Composition

Cereal lipids originate from membranes, organelles, and spherosomes and consist of different chemical structures. Depending on cereal species average lipid contents of 1.7–7% in the grains are present (Table 2.2). Lipids are mainly stored in the germ, to a smaller extent in the aleurone layer and to the lesser extent in the endosperm. In particular oats are rich in lipids (6–8%) in contrast to wheat and rye (1.7%). Cereal lipids have similar fatty acid compositions, in which linoleic acid reaches contents of 39–69%, while oleic acid and palmitic acid make up 11–36% and 18–28%, respectively [106, 107]. Although wheat lipids are only a minor constituent of the flour, they greatly impact the baking performance and have, therefore, been extensively studied. While triglycerides are the dominating lipid class in the germ and the aleurone layer, phospho- and glycolipids are present in the endosperm (Fig. 2.5). Depending on the extraction rate wheat flour contains 0.5–3% lipids [108]. Extraction with a polar solvent at ambient temperature, i.e., water-saturated butanol, dissolves the nonstarch lipids that make up approximately 75% of the total flour lipids [109]. The residual 25% are the so-called starch lipids. The composition of the nonstarch lipids is given in Table 2.8. They contain about 60% nonpolar lipids, 24% glycolipids, and 15% phospholipids. By extraction with solvents of different polarities they can be further subdivided into a free and a bound fraction. The nonpolar lipids are mainly present in the free lipid fraction, whereas glyco- and phospholipids are part of the bound fraction, in which they can be associated, for example with proteins [106, 107]. The major glycolipid class is the digalactosyldiglycerides. Starch lipids are primarily composed of lysophospholipids, which form inclusion complexes with amylose helices already in native starch [28].

2.4.2

Effects of Lipids on the Baking Performance of Wheat Flour

Only nonstarch lipids affect the rheological properties of wheat doughs. Interactions between starch lipids and starch are sufficiently strong so that this lipid fraction is not available before the starch gelatinizes. Studies with nonstarch lipids have shown that only the polar lipids have a positive effect on baking performance, whereas the

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Fig. 2.5 Polar lipids that affect the baking performance of wheat

Table 2.8 Composition of nonstarch lipids of wheat flour [107]. Content (g/100 g) based on total lipid Nonstarch lipids: 1.70–1.95 g/100 g flour Polar 36–42 Nonpolar 58–64 Phospholipids 14–16 Sterol esters 1.9–4.2 Acylphosphatidyl ethanolamine 4.2–4.9 Triglycerides 39.5–49.4 Acyllysophosphatidyl ethanolamine 1.6–2.3 Diglycerides 3.3–5.4 Phosphatidyl ethanolamine/phosphatidyl 0.7–1.1 Esterified monogalactosyldi2.7–3.9 glycerol glycerides/monoglycerides Phosphatidyl choline 3.8–4.9 Esterified sterolglycerides 0.8–4.2 Phosphatidyl serine/phosphatidyl inosit 0.4–0.7 Lysophosphatidyl ethanolamin 0.3–0.5 Lysophosphatidyl glycerol 0.2–0.3 Lysophosphatidyl choline 1.4–2.1 Glycolipids 22–26 Monogalactosyldiglycerides 5.0–5.9 Monogalactosylmonoglycerides 0.9–0.4 Digalactosyldiglycerides 12.6–16.5 Digalactosylmonoglycerides 0.6–3.4

nonpolar lipids have the opposite effect [110]. In particular glycolipids have been shown to contribute to the high baking performance of wheat flour [29, 111–113], whereas the functionality of the phospholipids has been found to be less important. If the term “specific baking activity” would be defined, polar lipids would be found to affect the baking performance of wheat flour to a considerably greater extent than proteins. The addition of only 0.13% polar lipids would yield the same increase of loaf volume as a protein content that would be increased by 1%. Polar lipids affect dough properties in many ways, i.e., the dough handling properties are improved

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and the gas-holding capacity during proofing is increased enabling a prolonged oven spring, increased loaf volume, better crumb resilience, and, in some cases, retardation of bread staling.

2.4.3

Modes of Action of Polar Lipids in Baking

The high baking activities of polar lipids, in particular of the glycolipids, might be explained by modes of action based on the formation of liquid films at the dough liquor/gas cell interface. Possible modes of action are the direct influence of the surfactants on the liquid film lamellae and gas cell interfaces through direct adsorption resulting in an increase of surface activity as suggested by Gan et al. [49, 114] and Sroan et al. [115] as the secondary stabilizing mechanism in the so-called dual film theory. It suggests the presence of liquid lamellae, providing an independent mechanism of gas cell stabilization. As shown recently, the effects of different surface active components may be explained by the type of monolayer that they form [116]. However, in particular the positive effect of some polar lipids such as acylated sterol glucosides and sterol glucosides cannot be explained with this mode of directly stabilizing the liquid film lamellae. Here another mode of action could be the answer, for example the indirect stabilization of the dough liquor/gas cell interface through this type of surfactant [116, 117]. These polar lipid classes have a positive influence on the phase behavior of the endogenous lipids present in the dough liquor in that they lead to an increase in surface activity of the endogenous lipids and hence a better availability and accumulation at the liquid film lamellae/gas cell interface, thus increasing gas cell stabilization, and consequently the bread volume. Inclusion complexes between amylose helices and polar lipids with one fatty acid residue are responsible for two effects. Complexes present in native starch (starch lipids) increase the temperature of gelatinization and, thus, prolong the oven spring. Inclusion complexes between amylose helices and polar lipids with one fatty acid residue may also form during and after the gelatinization process and are responsible for the anti-staling effect of some polar lipids, for example monoglycerides.

2.5 2.5.1

Minor Constituents Minerals

The mineral content of cereals ranges from ca. 1.0 to 2.5% (Table 2.2). Compared to other foods this is an intermediate concentration with milk, meat, and vegetables having somewhat lower mineral contents and pulses, which are extraordinarily rich in minerals (mean mineral content ~3.5%). As cereals are among the most important staple foods, and are consumed in high quantities, they are important sources of

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minerals in the human diet. The major portion of the minerals (>90%) is located in the outer layers of the grains, namely in the bran, the aleurone layer, and the germ. Consequently, products made from whole grains should increasingly be introduced into human nutrition to benefit from the mineral content of cereals.

2.5.2

Vitamins

Cereals contain vitamins in concentrations ranging from below 1 to ca. 50 mg/kg, depending on the compound (Table 2.2). Thus, cereals are a good source of vitamins from the B-group, and, in industrial countries, they cover about 50–60% of the daily requirement of B-vitamins. The most important fat-soluble vitamins are the tocopherols, which are present in concentrations exceeding 20 mg/kg. Like the minerals, vitamins are concentrated in the outer layers of the grains, in particular in the aleurone layer as well as in the germ. Therefore, milling of cereals into white flour will remove most of the vitamins. Consequently, the use of whole-grain products or products enriched in vitamin-containing tissues will be of nutritional benefit for the consumer.

Abbreviations AX DSC GMP GS HMW HPLC LMW m/a MMW MW MWD NSP PAGE SDS TG WEAX WUAX

Arabinoxylans Differential scanning calorimetry Glutenin macropolymer Glutenin subunits High-molecular-weight High-performance liquid chromatography Low-molecular-weight Monomeric/aggregated Medium-molecular-weight Molecular weight Molecular weight distribution Nonstarch polysaccharides Polyacrylamide gel electrophoresis Sodium dodecyl sulfate Transglutaminase Water-extractable arabinoxylans Water-unextractable arabinoxylans

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90. Hanft F, Koehler P (2005) Quantitation of dityrosine in wheat flour and dough by liquid chromatography-tandem mass spectrometry. J Agric Food Chem 53:2418–2423 91. Bauer N, Köhler P, Wieser H, Schieberle P (2003) Studies on the effects of microbial transglutaminase on gluten proteins of wheat. I. Biochemical analysis. Cereal Chem 80:781–786 92. Kasarda DD (1989) Glutenin structure in relation to wheat quality. In: Pomeranz Y (ed) Wheat is unique. AACC, St. Paul, pp 277–302 93. Tatham AS, Shewry PR (1985) The conformation of wheat gluten proteins. The secondary structures and thermal stabilities of a-, b-, g- and w-gliadins. J Cereal Sci 3:104–113 94. Müller S, Wieser H (1997) The location of disulphide bonds in monomeric g-type gliadins. J Cereal Sci 26:169–176 95. Tatham AS, Miflin BJ, Shewry PR (1985) The b-turn conformation in wheat gluten proteins: relationship to gluten elasticity. Cereal Chem 62:405–412 96. Esen A (1987) A proposed nomenclature for the alcohol-soluble proteins (zeins) of maize (zea-mays-l). J Cereal Sci 5:117–128 97. Wilson CM (1991) Multiple zeins from maize endosperms characterized by reversed-phase HPLC. Plant Physiol 95:777–786 98. Rubenstein I, Geraghty D (1986) The genetic organization of zein. In: Pomeranz Y (ed) Advances in cereal science and technology, vol VIII. AACC, St. Paul, pp 297–315 99. Shull JM, Watterson JJ, Kirleis AW (1991) Proposed nomenclature for the alcohol-soluble proteins (kafirins) of Sorghum bicolor (L. Moench) based on molecular weight, solubility, and structure. J Agric Food Chem 39:83–87 100. Watterson JJ, Shull JM, Kirleis AW (1993) Quantitation of a-, b-, and g-kafirins in vitreous and opaque endosperm of Sorghum bicolor. Cereal Chem 70:452–457 101. Hamaker BR, Mohamed AA, Habben JE, Huang CP, Larkins BA (1995) Efficient procedure for extracting maize and sorghum kernel proteins reveals higher prolamin contents than the conventional method. Cereal Chem 72:583–588 102. Danno G, Natake M (1980) Isolation of foxtail millet proteins and their subunit structure. Agric Biol Chem 44:913–918 103. Juliano BO (1985) Polysaccharides, proteins, and lipids. In: Juliano BO (ed) Rice chemistry and technology, 2nd edn. AACC, St. Paul, pp 98–142 104. Mandac BE, Juliano BO (1978) Properties of prolamin in mature and developing rice grain. Phytochem 17:611–614 105. Kruger JE, Reed G (1988) Enzymes and color. In: Pomeranz Y (ed) Wheat chemistry and technology, vol I, 3rd edn. AACC, St. Paul, pp 441–476 106. Delcour JA, Hoseney RC (2010) Principles of cereal science and technology, 3rd edn. AACC International, Inc, St. Paul, pp 40–85 107. Eliasson A-C, Larsson KA (1993) Molecular colloidal approach. In: Cereals in breadmaking. Marcel Dekker, Inc, New York 108. Hoseney RC (1994) Principles of cereal science and technology, 2nd edn. AACC, St. Paul, pp 81–101, and 229–273 109. MacMurray TA, Morrison WR (1970) Composition of wheat-flour lipids. J Sci Food Agric 21:520–528 110. Morrison WR, Mann DL, Soon W, Coventry AM (1975) Selective extraction and quantitative analysis of nonstarch and starch lipids from wheat flour. J Sci Food Agric 26:507–521 111. MacRitchie F (1981) Flour lipids: theoretical aspects and functional properties. Cereal Chem 58:156–158 112. Daftary RD, Pomeranz Y, Shogren M, Finney KF (1968) Functional bread-making properties of wheat flour. II. The role of flour lipid fractions in bread making. Food Technol 22:327–330 113. Hoseney RC, Pomeranz Y, Finney KF (1970) Functional (breadmaking) and biochemical properties of wheat flour components. VII. Petroleum ether-soluble lipoproteins of wheat flour. Cereal Chem 47:153–160 114. Gan Z, Angold RE, Williams MR, Ellis PR, Vaughan JG, Galliard T (1990) The microstructure and gas retention of bread dough. J Cereal Sci 12:15–24

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115. Sroan BS, Bean SR, MacRitchie F (2009) Mechanism of gas cell stabilization in bread making. I. The primary gluten-starch matrix. J Cereal Sci 49:32–40 116. Sroan BS, MacRitchie F (2009) Mechanism of gas cell stabilization in breadmaking. II. The secondary liquid lamellae. J Cereal Sci 49:41–46 117. Selmair PL, Koehler P (2009) Molecular structure and baking performance of individual glycolipid classes from lecithins. J Agric Food Chem 57:5597–5609

Chapter 3

Technology of Baked Goods Maria Ambrogina Pagani, Gabriella Bottega, and Manuela Mariotti

3.1

Introduction

Baked goods are a heterogeneous market category of products. The high diversification that distinguishes these products makes it difficult to find one single, general and satisfactory definition. It is, however, possible to identify baked goods based on the common commodities matrix since they are all foods derived from cereal flour [1]. Other similarities within this group are the basic ingredients used, mainly wheat flour or, less commonly, rye flour, water and leavening agents. Although the technological processes may differ, each one comprises mixing, leavening and baking. These successive stages allow the transformation of flour, or a mixture of different types of flour, into an appetizing and digestible food, which, at the macroscopic level, has a complex structure differentiated by a friable crust and an internal alveolar structure. The classification of baked goods may be based on different criteria [2]. One criterion, which is widely used in the baking trade but is not governed by legislation, is the presence of sugar in the formula which can be perceived by taste and corresponds to at least 10% of the weight of the flour. Baked products with more simple formulas, therefore, constitute the various types of bread. Despite its importance, not only sensorial but also nutritional and textural, the amount of sugar in the dough is not sufficient for classifying this complex category in a satisfactory way. As proposed by Cauvain and Young [1], the distinction of the basic products comprising the formula may be made by considering the fat:flour ratio.

M.A. Pagani (*) • G. Bottega • M. Mariotti (*) Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, via Celoria 2, 20133 MILANO, Italy e-mail: [email protected]; [email protected]; [email protected] M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, DOI 10.1007/978-1-4614-5425-0_3, © Springer Science+Business Media New York 2013

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A further criterion for differentiating baked goods is the evaluation of their lightness and softness, and so of the specific volume (correlated with lightness) and humidity (which determines the softness). These two structural characteristics are the result of complex phenomena that occur during each stage of the technological process. Indeed, the final characteristics of the product not only depend on leavening but also on mixing and baking (see Sect. 3.4). A light and soft baked good usually has a specific volume higher than 2.5–3.0 mL/g and humidity higher than 15–18%. A dry and friable baked good has a specific volume between 1.3 and 2.5 mL/g and humidity values lower than 5–10% [2]. On the basis of these two criteria, four categories of baked products can be identified. Moreover, each category can be further subdivided according to the leavening method used (see Sect. 3.3.3).

3.2 Wheat, a Preferred Raw Material for Bread and Baked Goods The choice of ingredients is of fundamental importance for the production of leavened baked goods that satisfy consumer tastes. Although barley and rye flours provide a workable dough, bakers realized a long time ago that the best results in terms of volume development were always obtained with wheat flour. The superior quality of this cereal, especially its most evolved species Triticum durum and T. aestivum, is of a purely technological nature. Only wheat flour provides a cohesive and homogenous dough, where the single and original particles of flour are no longer recognizable. The property that makes wheat dough unique is its viscoelasticity. This rheological property enables the mass to be stretched and deformed without rupturing. At the same time, the dough is elastic and tenacious, capable of maintaining its shape even when subjected to physical stress. After baking, gluten proteins denature and loose viscoelasticity, ensuring the maintenance of the final shape of baked goods. The reason for this versatile behaviour does not depend on differences in the quantity of components. Indeed, the protein content of the numerous varieties of wheat extends over quite a wide interval, from 9 to 16% of the weight of the grain [3]. This variability coincides with that of other cereals. The technological superiority of wheat is related to complex qualitative differences at the level of protein fractions. Wheat storage proteins are generally classified based on molecular weight (MW), solubility and conformation. Storage proteins, gliadins and glutenins, account for ca. 80% of the entire protein fraction and have a particular amino acid composition. Gluten proteins have a high percentage of glutamine, about one third of all amino acids, and proline, and low levels of lysine (Chap. 2, [4]). This composition is responsible for the low nutritional value of wheat protein. However, it also accounts for the protein–protein interactions that lead to the formation of gluten, the threedimensional network which is continuous and homogenous throughout the mass. Both non-covalent bonds, such as hydrophobic interactions and hydrogen bonds (guaranteed by the large amounts of glutamine), and covalent bonds are involved, the most significant of which are the disulfide bonds between cysteine residues.

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Gliadins and glutenins have different roles for the viscoelastic characteristics of gluten [5–7]. Gliadins can be easily deformed and stretched, thanks to their viscous properties that are typical of fluids, because of their globular shape and interconnections via disulfide bonds. In contrast, glutenins form long chains by means of interprotein disulfide bonds, which resist deformation and form an elastic and tenacious mass. Although durum wheat semolina is used for bread making, especially in the Mediterranean regions, the common wheat (T. aestivum) flour may be the optimal raw material because it gives the best leavening results [8]. Soluble proteins in wheat flour are mostly proteins with enzymatic activity. Most of these enzymes are hydrolases (amylases, proteases, lipases), which specifically act on the reserve macromolecules. Also oxidative enzymes (lipoxygenases, peroxidases) are present. For many wheat processes, the enzymatic activities have a central and strategic role which has to be carefully monitored (see also Chap. 2).

3.2.1

The Milling Process

About 70% of the worldwide wheat production is used for food [9], mainly by bakery industries. The essential preliminary step is the milling process. The objective of milling is twice. On the one hand, it separates the starchy endosperm, the area containing gluten proteins, from germ, pericarp and seed coats, that form bran and other by-products. On the other hand, it reduces the particle size of the endosperm to values lower than 150–200 mm for common wheat flour and 500 mm for durum wheat semolina. The fine granulometry of the flour gives an optimal workability to the resulting dough and contributes to its processing into a palatable and appetizing food. The removal of bran improves both the hygienic properties of the flour, since the peripheral parts of the grain are often contaminated by chemical residues and biotic pollutants, and the technological characteristics of the flour. In fact, nonstarch polysaccharides and enzymes, which are abundant in the bran, worsen the rheology properties of the dough [10]. The separation of the oil-rich germ prevents rancidity, which compromises the storage of flour [11]. However, the elimination of these two parts, which are rich in several functional components, decreases the nutritional value of the flour. Overall, milling is much more complex than mere grinding. Because of the particular morphological structure of wheat grain, which is characterized by the crease or ventral furrow, an introflexion along the length of the kernel, which hides a double layer of teguments, the milling has to promote the extraction of the flour from grains. Therefore, the first step consists of opening the grain. Then, proceeding from the inside towards the outside, the endosperm is recovered via repeated sequences of size reduction and separation stages, excluding the more external areas (bran, aleurone layer, etc.), which are known as tailing products. This procedure fully justifies the definition of “flour extraction yield”, defined as the quantity of flour produced from 100 parts of cleaned and conditioned wheat grains [12]. This is the only solution for preventing the passage of the bran layers from the furrow, in which

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Table 3.1 Chemical composition of wheat regions (Adapted from [15]) (data expressed as g/100 g dm) Kernel Starch and soluble Proteins Lipids NSPa (cellulose, Region (%) sugars (%) (%) (%) etc) (%) Ash (%) Fruit coat 5 14 ÷ 16 10 ÷ 14 1÷3 60 ÷ 74 3÷5 (pericarp) Seed coat 2 9 ÷ 11 13 ÷ 19 3÷5 53 ÷ 63 9 ÷ 15 Aleurone 8 10 ÷ 14 29 ÷ 35 7÷9 35 ÷ 41 5 ÷ 15 Endosperm 82 80 ÷ 85 8 ÷ 14 2÷3 1÷3 0.5 ÷ 1.5 Germ 3 19 ÷ 21 36 ÷ 40 13 ÷ 17 20 ÷ 24 4÷6 a Non-starch polysaccharides

25–30% of total bran is hidden, into the flour of the bran layers [13]. Milling is generally simpler for those cereals without furrow. The physical separation of the various parts of the wheat grain is made possible by the different composition of the three morphological areas of the kernel, that determine different behaviour during processing (Table 3.1). The separation of the endosperm from the bran is not quantitative, since parts of the endosperm are lost into the milling by-products and small percentages of bran fragments are inevitably present in the flour. Therefore, the milling process has to reach a compromise between “extraction yield” and “grade of refinement” (accuracy of elimination of bran) of the flour. The main criterion for flour classification is based on the accuracy of teguments separation. The approach, used by legislation in many countries, is based on the threshold value of a number of parameters, especially ash and proteins. The current technology for milling considers the following four stages: (1) receiving, pre-cleaning and storage of the incoming wheat; (2) cleaning and conditioning; (3) milling; (4) storage of the flours. Wheat coming to the mill is usually transported in bulk in trucks, trains or ships and it is unloaded into large hoppers with a grilled opening to facilitate the elimination of large foreign matter. This operation is preceded by rapid analytical inspections of samples representative of the entire batch, to assess quality parameters. Preliminary cleaning operations made before loading the wheat into the silos are intended to remove mainly coarse foreign materials, in order to provide for better storage. The cleaning of the wheat is carried out immediately before the milling process. This involves a sequence of operations, each performed by a special machine, with the aim of removing impurities, foreign matters and powders. Differences in size, shape and density compared to the whole and sound wheat grains are used to achieve this aim [14]. The conditioning or tempering of kernels is decisive to achieve an optimal milling. This operation includes grain humidification and the successive resting time, to increase the water content of 2–4%. The conditioning step toughens the bran, favouring its break off in form of large particles, and mellows the endosperm, thereby facilitating the separation between these two parts. The conditioning time (from 6 to 24–36 h), the quantity of water used, and the ways to add water (one or two tempering steps) depend on the initial humidity and on the hardness and vitreousness of the wheat kernels.

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Fig. 3.1 Simplified schema of wheat milling operations (Adapted from [15])

After this stage, caryopses have 15.5–17% humidity and are in the best state for milling [14]. The milling process is complex in terms of both the type and number of operations involved and the methods used (Fig. 3.1). The process is classified into different systems: (a) break system, which separates the endosperm from bran and germ; (b) sizing or purification system, which separates particles according to the presence of bran pieces; and (c) reduction system, where the large particles of endosperm are reduced to flour [15]. First, the kernel is subjected to breaking by means of roller mills that are made up of pairs of cast iron cylinders, each at a set distance from each other, with corrugated surfaces and turning at differential speeds. The breaking system has the function to open, cut and flake the grains: it separates the endosperm from teguments and leaves bran in the form of large and flat flakes to facilitate their removal. Grains have to be ground gradually (four to five subsequent breaking steps), thus limiting the formation of flour and the disintegration of bran [12]. Each breaking step is followed by sieving, carried out by plansichters or sifters. They are large machines, within which numerous sieves are stacked with a mesh granulometry suitable for the material to be sifted (Fig. 3.2). The coarse particles of endosperm are called “semolina”. Some of them, referred to as middlings, have various degrees of attached bran layers, whereas others are clean middlings, composed of pure endosperm. These two fractions are separated according to their specific gravity and size. The sizing rolls, formed by smooth or slightly corrugated rolls, detach the bran pieces attached to the middlings. These operations are immediately followed by classification through plansichters. The clean middlings are sent

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Fig. 3.2 Sieving or grading section: plansichters (above) and middling fractions separated by sieving action (below) (Courtesy of Bühler AG, Switzerland)

to the reduction system, the final stage of the milling process, with the objective to reduce the size of clean middlings to flour. It consists of a sequence of several smooth roll mills (up to eight to ten, according to the size and the expected starch damage) and sifters. The milling diagram comprises a number of the above steps, to ensure that the majority of the endosperm is converted into flour and that most of the teguments are removed as by-products. The flour extraction yield varies between 74 and 76%. Since the bran and the germ together represent around 20% of the weight of the wheat grain, the flour extraction yield is lower than the theoretical value. More refined flours have lower extraction rates, since most of the external layers of the endosperm are eliminated with teguments. Milling of durum wheat requires different diagrams, which are characterized by a higher number of purifiers to improve the separation of bran particles. Nevertheless, the yield is lower (68–72%), since semolina is mostly formed by particles larger than 250–300 mm and contains only a minimum amount of fine particles (lower than 200 mm). The separation of the more external layers and germ of the caryopsis inevitably causes a marked change of the chemical composition and, consequently, of the nutritional value of the flour. It has a lower concentration of ash, proteins, vitamins and soluble sugars than the caryopsis and a higher starch content. This difference depends on the efficiency of the separation of the more external layers of the caryopsis from the endosperm. Consequently, refined flours, corresponding to an extraction yield of approximately 75%, contain only 5% of fibre, 45% of fats, 30% of

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Fig. 3.3 Composition of flour according to the extraction rate: comparison with whole kernel (dotted line refers to wheat kernel) (Adapted from [15])

minerals and between 15 and 40% of different vitamins in comparison to the native caryopsis (Fig. 3.3).

3.2.1.1

Milling Optimization and Innovation

During the last decade, new food safety-related regulations, for example HACCP, ISO Standards 9001; 22000 and 22005, traceability, labelling health claim and use of GMO, (FAO/WHO, 1997) have supported the improvement of the process control in milling and bakery industries [16, 17]. In particular, technological solutions that not only consider the production and economic aspects, but also the hygiene and nutritional characteristics of flour were proposed.

Cleaning The most recent innovation proposed for this stage is the use of an optical sorter (www.buhlergroup.com), a device that efficiently removes all types of contaminants and foreign materials in one step. The product stream is fed inside the sorting machine and different high-resolution cameras detect and recognize defects based on colour, shape and other optical properties. Specific sensors and high-speed ejectors

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carry out a precise ejection, enabling the accurate separation of contaminants in a very short time.

Debranning and Pearling Debranning technology, also referred to as pre-processing or pearling, is currently used in rice milling and barley processing for removing hulls that have no technological and nutritional value. Although this practice is necessary in the case of covered cereals, the sequential removal of the outer bran layers prior to milling is quite uncommon in naked grain processing. The original debranning systems for wheat are the Tkac process and the PeriTec process marketed by Satake Corporation. In both processes, two distinct machines assure the bran removal by friction (kernel to kernel) and abrasion (kernel to stone) actions [18, 19]. New debranning equipments for wheat were developed to carry out abrasion and friction on the same machine. The Vertical Debranner VCW (Satake Corporation; www.satake-group.com) includes two separate working chambers in the same equipment: the upper one has an abrasive zone where rotating abrasive rings work the grain against a peripheral slotted screen to eliminate the outer bran layers; partially debranned grains then enter the lower chamber, where friction completes the debranning process. Applying the Tkac system to several durum wheat varieties, Dexter et al. [20] found an increase in semolina yield and a higher semolina refinement. Consequently, the colour of pasta was improved. Other interesting results were also found at laboratory and industrial scales [21]. Because of the positive results obtained with durum wheat, attention was shifted to common wheat. Except for the decrease of the microbial contaminations [22] and bunt infections [23], the results are still contradictory. Currently, the pre-milling treatment of common wheat consists of a peeling process carried out with a machine (DC-Peeler Bühler AG – www.buhlergroup.com or the DHA Vertical Debranner from Ocrim S.p.A.—www.ocrim.it) which promotes a mild removal of the outermost layer of the kernel (maximum 1.5–2% of the grain weight) and, at the same time, enables one to reduce contaminations (bacteria, mycotoxins and heavy metals) by 50%.

3.2.2

Improvement of Flour Performance

The natural aptitude of wheat to be processed into varieties of baked goods is further improved by selective breeding research. These studies mainly concern changes of the quality/quantity of seed storage proteins. Nonetheless, the performance of wheat during processing is sometimes different from expectations due to characteristics that are markedly influenced by environmental and agronomic parameters [24]. The practice of improving the technological functionality of the flours by the addition of improvers is, therefore, significantly widespread. Among the most commonly used improvers there are enzymes (e.g. amylase, hemicellulase, lipase and protease) and

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emulsifiers (e.g. mono- and di-glycerides), which increase the volume of the dough and the gas retention and crumb softness of baked goods. It is interesting to note that many of these improvers are naturally present in the wheat grain but they are removed with the by-products (bran and germ) during milling.

3.3 3.3.1

Other Raw Materials Water

Water plays a key role both during processing and for the shelf-life and sensory properties of baked goods. On the basis of the definition used for studying polymers, the adjective “plasticizing” is often used for water. This identifies a material incorporated into a polymer to increase workability, flexibility and extensibility [25]. The water is subjected to significant changes during processing, both in terms of absolute quantity (total humidity) and availability (relative humidity). The amount of water added to convert flour to dough has to ensure the hydration of all hydrophilic components, especially proteins. The addition of this solvent determines the radical change of the three-dimensional conformation of proteins. In 1973, following their observations with an optic microscope, Bernardin and Kasarda described an “explosion” of flour particles when in contact with excess water, and the rapid formation of protein strings [26]. This spontaneous rearrangement is caused by the immediate exposure to water of the hydrophilic areas of proteins which are rich in polar amino acids, while the more hydrophobic areas are hidden inside. The interaction among different protein chains is ensured through the formation of disulfide bonds and through stabilization via hydrogen bonds and hydrophobic interactions. Gluten proteins undergo a kind of glass transition when they absorb water, passing from the hard and glassy state to the soft and rubbery state [27]. Cuq et al. [28] described the changes that occur during the bread making using state diagrams and phase changes. The glass transition is the period of marked increase in molecular mobility that involves amorphous polymers (e.g. proteins) or the amorphous areas of semi-crystalline polymers (e.g. starch). Amorphous polymers are in the glass state at low temperatures and/or with low water content. An increase of temperature or of the water content induces amorphous polymers to become soft and viscoelastic. When the water content is higher than 15–20%, the glass transition of proteins occurs below environmental temperature [28]. During mechanical mixing, the relative mobility of the protein molecules and their high reactivity cause the formation of intermolecular covalent links that are responsible for the formation of the continuous and homogenous gluten network. The level of water needed to obtain an optimal consistency of the dough is not always easy to quantify. Overall, hydration less than 35% does not give an optimal and homogenous hydration of gluten [29]. The absorption of water varies according to the degree of refinement, granulometry, level of damaged starch, quantity and quality

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of proteins and humidity of the flour. The hydration capacity of the flour is usually calculated based on the farinographic absorption index (see Sect. 3.5.1.1). Although it is very practical for routine applications, this approach does not describe the distribution of water within the components and the competition among the hydrophilic compounds. The dough is a highly complex system where numerous aqueous stages coexist and each one is variously rich in chemical components [30]. The dough is a sophisticated metastable dispersed system, where water moves from one phase to another and where thermodynamic incompatibility among different polymers occurs [31]. The time needed to obtain homogeneous and uniform hydration of the flour particles is strictly related to the dimension of the particles, their hardness and vitreosity, the presence of non-starch polysaccharides and intensity of mixing. Water is not only necessary for the formation of gluten, but it also performs a solvent action for the other ingredients present in the formula (e.g. salt and sugars) and it allows the enzymatic activities to take place. Another function played indirectly by water is the control of the dough temperature. After mixing, ca. 45% of the total water in dough is associated with starch, 30% with proteins and 25% with non starch polysaccharides (pentosans) [32]. Water allows the swelling of the starch granules during baking and their gelatinization, a key phenomenon for the physical and nutritional properties of baked goods [33].

3.3.2

Other Ingredients: Salt, Sugar and Fats

Flour, water and leavening agents are the indispensable ingredients for making baked goods. Often the formula requires the addition of salt, which influences the sensory and rheological properties of the dough, and additives or improvers. The use of salt in leavened baked products generally refers to sodium chloride. Salt is an ingredient that is almost always present in the formulation of bread and other bakery products. The role of salt is related to its ability to enhance the aroma of the product and to mask off-flavours such as a bitter and metallic taste, but salt addition also strengthens the structure of the dough. This effect on dough structure is caused by the positive effects on both hydrogen bonds and hydrophobic interactions between protein macromolecules. Salt optimizes the mixing time and the kneading, and controls the speed of yeast fermentation [34]. In dough without salt, the high speed of CO2 production may be responsible for the deterioration in the product structure. On the contrary, NaCl allows the leavening step to be controlled and optimized by slowing the rate of gas production [35]. However, this positive effect is strongly influenced by the amount of salt added [36]. In particular, a great increase in volume was observed in bread prepared by adding 1.5–2% of NaCl, while quantities exceeding this threshold were associated with a strong decrease in the volume of the product. Other ingredients such as sugar or fats are optional. The addition of sugar or fat over a certain value markedly changes the rheological properties of the dough. The presence of sugars influences each stage of processing: it gives more consistency to the dough, in some cases promotes the fermentation, facilitates the browning of the

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crust during baking and ensures a soft crumb during storage. Fats such as olive oil, lard or butter are also used. If the concentration of fat is lower than 5%, the main technological role is as a lubricant: the gluten increases the extensibility before rupture, favouring a higher dough volume [37]. If the formulation is very rich in fat, the dough becomes short and loses its extensibility. This effect justifies the definition of shortening for these components. Polar lipids such as mono- and di-glycerides stabilize the air bubbles formed during mixing and provide a crumb with a finer and more regular alveolar structure [38]. During storage, lipids prevent the interaction among starch macromolecules, slowing down their reorganization into ordered and crystalline structures (retrogradation), as well as the migration of water between starch and proteins, slowing down the phenomena of staling and aging [39].

3.3.3

Leavening Agents

Essentially three types of leavening agents are used for making baked goods: baker’s yeast, chemical agents and sourdough. Since sourdough will be covered in detail in all the other chapters, this paragraph only aims at shortly describing the main features of the other leavening agents.

3.3.3.1

Baker’s Yeast

Baker’s yeast refers to Saccharomyces cerevisiae strains. After a brief initial respiratory activity due to the oxygen dispersed in the dough, baker’s yeast ferments glucose, fructose, maltose and sucrose from the flour to CO2 and ethanol. Nowadays, the types of baker’s yeast available on the market have different shelf life (yeast cream, compressed yeast, dried yeast), osmotolerance features (suitable for baked goods with elevated levels of sugars) and activity at low temperatures (frozen dough) (see Chap. 6). The use of baker’s yeast as a leavening agent is very often an alternative to the use of sourdough, especially for industrial bakeries. During processing, baker’s yeast mainly determines the leavening of the dough due to the production of CO2; it also synthesizes some volatile compounds that positively affect the flavour and taste of baked goods.

3.3.3.2

Chemical Agents

The production of CO2 within the dough can also be obtained by a reaction between sodium bicarbonate and an acid (e.g. tartaric acid). Chemical agents are not used for bread making but for sweet baked goods, both dry and light (such as biscuits, spongecakes, etc.). The use of chemical agents for leavening is recommended when CO2 has to be produced rapidly in doughs rich in sugars and fats, ingredients that slow down and/or inhibit the metabolism of the biological agents. Sodium bicarbonate lacks

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toxicity, is cheap and easy to use, and it has a high solubility at room temperature. On the basis of the rapidity of the reaction, the chemical agents are classified as: fast or immediate, slow or delayed, and double acting powders. The former develop gas as soon as they are introduced into the dough. The delayed acting powders determine the formation of negligible quantities of gas during mixing, which only develops at high temperatures during baking in the oven. The double effect powders react in part at room temperature and in part during baking, and in this case two acids are involved, one soluble and one insoluble. The reactivity of a chemical agent is expressed as the “neutralization value” (g of NaOH that are neutralized by 100 g of acid salt).

3.4

Baked Goods Making Process

A very large number of baked goods are manufactured worldwide. Breads are the most diverse and several differences are found, for instance, between those manufactured in the Mediterranean areas and those from the Anglo-Saxon market [40]. Overall, in the Mediterranean areas no significant quantities of sugar or other high hydrophilic substances are added and pans are generally not used in the leavening and baking stages. Apart from the large variety of baked goods, the technological process may be summarized in a sequence of operations that require long periods of time and which have the primary objective of aerating the dough and making it porous (Table 3.2).

3.4.1

Discontinuous Processes (Straight-Dough and Spongeand-Dough)

Bread making, both at artisan and industrial levels, is traditionally a discontinuous process since the various stages of mixing, leavening and baking are carried out on limited quantities of materials and in separated facilities. Discontinuous bread-making processes are performed using the straight-dough or the sponge-and-dough methods. Bread making with sourdough could be considered as a particular sponge-anddough method. Bread characteristics are influenced not only by processing but also by flour and formula. In the straight-dough method, the fastest and easiest to manage, all the ingredients are mixed together simultaneously to form a dough which is then left to rise (Table 3.3). Fermentation has to be carried out at least in two stages. The first leavening is generally made with large quantities of dough, and for variable times (from 30 min to 3 h), depending on the process. The primary objective of this operation is not to get a volume increase, but to induce changes in the rheological properties of the dough [41]. The first leavening provides a greater workability to the dough, which achieves the capacity to maintain shape during the second leavening (or proofing). In this stage, individual pieces of dough, corresponding to the final size,

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Table 3.2 Bread-making process: aim and modifications associated with the main operations Step/phase/operation Aim Modifications Hydration and solubilization of Mixing Homogeneous distribution of the water compounds ingredients (including minor components) Formation of uniform and Formation of soluble gluten “coherent” structure Inclusion of air bubbles Inclusion of air microbubbles Leavening/proofing Increase in volume of dough Formation of gas (CO2) Development of typical flavour Production of fermentation metabolites important for developing flavour and able to change the macromolecules solubility Shaping Giving shape to dough Subdivision of gas bubbles and inclusion of new air Division of dough into final pieces Cooking Giving the product its typical aspect Increase in volume due to evaporation of gases: 20–30% of the volume is obtained during baking (oven-spring) Formation of crust and crumb Decrease of water content Protein denaturation Stabilization of leavened and shaped Starch gelatinization dough Making product appetizing and Development of flavour digestible Completing leavening of the dough Evaporation of water and ethanol Cooling Product packaging Change of solubility of sugars Hardening of fats

Table 3.3 Characteristics of bread obtained according to the nature of the flour (straight-dough process using 100 g flour) Bread Specific volume Cereal flour bread Volume (mL) Height (mm) Weight (g) (mL/g) 100% wheat flour 100% einkorn flour 70% wheat flour + 30% rye flour 70% wheat flour + 30% maize flour

900 440 485

121 74 85

140 142 148

6,4 3,0 3,3

440

75

146

3,0

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are maintained at controlled temperature and humidity conditions for approximately 1 h until the maximum volume is reached. With the sponge-and-dough method, the ingredients are added at different times, during the refreshments of the dough. Using baker’s yeast, the sponge starts by mixing compressed yeast with part of the flour and water (from 50 to 70% of the total dough) required for the formula. After a leavening phase (from 3 to 4 h up to 10 h or more), the remaining parts of flour and water are added, together with any other ingredient required. During the maturing stage, the yeast adapts to the environment and reaches the optimal fermentative capacities when all the ingredients are added. The final dough is cut into pieces, shaped and left to rise again for about 1–2 h, before being baked. The long leavening time required to get the sponge ensures an alveolar structure with a large number of bubbles in the product, some of them of considerable size [42]. This structure guarantees extreme lightness, as indicated by the high specific volume and porosity of the crumb (Table 3.4). According to the consistency of the sponge, different definitions of the process are given. Polish, Poolish or Viennese methods use highly fluid dough. This process was initially introduced in Poland and subsequently widespread in Vienna at the beginning of the last century [41]. The Poolish premix is generally prepared using 50–75% of the water required by the recipe, to which an equivalent amount of flour is added. This ratio gives a mass of low consistency that is left to ferment from 3 to 8 h, according to the quantity of baker’s yeast added. This method gives sensory advantages and allows the formation of a delicate aroma. Leavening with sourdough requires the use of a starter, represented by a piece of dough from a previous batch which is fermented and stored under controlled conditions of temperature and humidity. Sourdough is generally used at 5–20% of the formula. Refreshing is of fundamental importance for dough development and maintains the microbial species typical of the sourdough [43, 44]. The intense acidification markedly influences the sensory and shelf-life features of the baked goods [43]. In particular, making bread with sponge and dough methods, and especially using sour dough, ensures a higher initial lightness compared to the straight dough method (Table 3.4). The bread is characterized by a more highly developed and irregular alveolar structure, since the longer fermentation times determine a slow and progressive production of CO2, accompanied by coalescence phenomena. The bread also has a characteristic taste and smell, caused by the formation of volatile compounds that are mainly formed during baking, following the Maillard reaction between glucose and free amino acids, which are released during fermentation. In Anglo-Saxon countries, and in particular in Great Britain, the most widespread bread-making process is the straight dough method known as the Chorleywood Bread Process (CBP). It was developed in the 1960s by the Flour Milling and Baking Research Association at Chorleywood in England [38, 45]. The leavening period is substantially reduced thanks to the high-speed mixers used for mixing the dough (see Sect. 3.4.3.1). CBP uses an intense mechanical mixing of the dough that lasts just a few minutes and ensures the incorporation of large quantities of air inside the mass. The decrease of time limits the cost of the process but the special conditions used during mixing require enriched flours and formulas with elevated

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concentration of baker’s yeast (higher than 2.5–3.0%). Emulsifiers and fats with a high fusion point are also indispensable for the oxidizing properties and to stabilize the numerous tiny alveoli that develop from the microscopic air bubbles included during the mixing stage. Another characteristic of this process is the reduction of the pressure during mixing to adjust the size of the alveoli.

3.4.2

Continuous Processes

These processes were introduced in the USA during 1950. The two best-known types are the Do-Maker process, developed in 1954, and the Amflow process, developed in 1960 by AMF Incorporated [46]. These technologies took hold in the 1970s, and were widespread especially in the United States and Great Britain. Contrarily to the discontinuous process working in batches, the continuous process is characterized by a substantial reduction of time, more compact machinery and better durability of the characteristics of the baked goods [47]. These processes are based on the possibility of eliminating the long leavening times by using yeast cultures or pre-ferments propagated separately without or with small quantities of flour. The subsequent high-speed mixing, with the simultaneous addition of all the ingredients, favours volume development even without long leavening times. As for the Chorleywood process, the intense mechanical stress during the high-speed mixing can be “supported” by the dough only if strong oxidizing improvers are added; emulsifying lipids are also indispensable.

3.4.3

The Main Stages of the Process

3.4.3.1

Mixing

As shown in Table 3.2, during mixing ingredients are distributed and blended within the mass, and gluten is formed. These phenomena are described as dough development. Many variables are involved. One of these is the quantity of water added to the flour, which may be indicated as the “level of absorption” or “hydration”. In some processes the level of absorption does not correspond to the optimal quantity as determined by the farinograph but it is mainly related to the handling characteristics of the dough. Stiff dough with hydration levels between 40 and 45% (dough humidity: 38–41%) has reduced extensibility; consequently, the baked goods have a limited porosity with a very fine alveolar structure. Soft or slack doughs have hydration levels higher than 60% (dough humidity: about 50%). They are difficult to handle due to their low consistency, which is responsible for the long and irregular shape such as the Italian Ciabatta [2]; the crumb presents large alveoli, often long in shape, which result from the coalescence of smaller bubbles.

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Table 3.4 Physical characteristics of flour, dough and bread according to the bread-making process Specific volume (mLg−1) Flour 1.5 Dough 0.85 Proofed dough 0.9–1.0 Wheat bread – Straight 2.5 ÷ 3.0 and dough

– Sponge and dough

Porosity (%) – – – 20 ÷ 25

3.0 ÷ 10.0a 25 ÷ 75a

– Sourdough 3.2 ÷ 3.7

35 ÷ 40

a

According to the parameters of the technological process

Mixing during traditional processes is carried out at least in two different times with different speeds. In the first stage (the French frasage), the low speed lasts about 5 min. Water is distributed among ingredients to allow the hydration of the protein macromolecules and the development of gluten. Air bubbles are entrapped into the mass and their sizes vary between 30 and 100 mm [48]. This phenomenon is completed during the high-speed stage (the French malaxage and soufflage). At the microscopic level, the sequence of events is associated with significant rheological changes and the mass is rather wet and sticky until hydration is completed during the cleanup stage (Fig. 3.4). Development of the dough is completed when the mass clears away from the walls and blades of the mixer bowl and starts to crackle in the bowl. Furthermore, stretching deforms the dough without breaking until it becomes a semi-transparent film [47], thanks to the viscoelastic properties of the gluten.

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Fig. 3.4 Raw materials and “evolution” of dough aspect during dough-making

The air included at the end of the mixing stage represents ca. 8–10% of the volume of the dough [48]. From this point onwards, any further mixing causes irreversible changes of the rheological properties. The over-mixed dough becomes sticky and loses elasticity.

The Mixers The mixers are usually classified into two categories: vertical and horizontal mixers. The former consist of machines with rotating bowls of various capacities ranging from 100 to 3,000 kg and more. The first machines, known as Artofex, have two reciprocating arms whose movement, both circular and vertical rotation at moderate speed, simulates the movement of the baker’s arms. The action performed by the dual-arms is very delicate. Consequently, the time necessary to completely develop the dough is long and the productivity of the machine is low. Currently, they are almost completely substituted by spiral mixers. The mechanical action against the dough is usually completed by a rotating central post, a device whose function is to hold and expose the dough to the action of the spiral tool, decreasing cutting effects with a smoothly mixing action. The different types of mixer vary depending on the capacity and rotation speed of the bowl, the arrangement and rotation speed of the mixing tools, the possibility of cooling down the mass via insufflation of CO2 and the possibility of extracting the bowl to guarantee the easy movement within the working premises. As mentioned previously, high-speed mixers, capable of completing the process in a few minutes and ensure the retention of large volumes of air, were developed to increase the productivity of the bread-making process. The horizontal bowl, normally made of stainless steel, presents a single mixing shaft with several transversal bars, whose profile varies in function of the process involved. Auxiliary equipment includes the microprocessor controls for monitoring all mixer functions. These devices ensure constant and uniform characteristics in all dough batches. In the Chorleywood method, the Tweedy mixers are preferred (www.bakerperkinsgroup. com). These high-intensity mixers have an impact mixer plate at the bottom of the bowl. Some baffles are present at the sidewalls of the bowl to direct the dough towards the mixer plate. The rotation speed of the mixing device may exceed 300 rpm, while the bowl remains fixed. These conditions are suitable to fully develop dough in only 4–5 min.

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Fig. 3.5 Planetary mixer and examples of mixing tools (Courtesy of Sancassiano S.p.A., Italy)

The quantity of air retained in the dough varies based on the mixer characteristics, flour strength and the addition of specific ingredients. To obtain batters (highly aerated dough for sugar- and fat-based cakes), the planetary mixers are used. Their mixing tool is similar to a whisk (Fig. 3.5) and performs a whipping action on the mass. The presence of emulsifiers is indispensable to stabilize this very low density foamy mass and to produce an extremely fine grain and uniform texture. According to the mixing tool shape and the speed applied, the time needed to obtain a well-developed dough or a light batter varies from 30 to just a few minutes. Consequently, the temperature of the mass remains the same or just a little higher

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Fig. 3.6 Carousel system (Courtesy of Sancassiano S.p.A., Italy)

than that of the ingredients (mixers with dual arms or spirals, 20–60 rpm) or may increase even by 10–12°C in the case of ultra-high-speed mixers, working at 500–1,000 rpm. The mixers previously described work discontinuously. The developed dough must be removed and the bowl emptied ready for the next batch. This system has a limited effect on the organization of the work at artisanal level but at industrial plants it creates serious problems. One proposed solution consisted of the carousel, a modular system which, although it did not shorten the mixing times, guaranteed the availability of a well-developed dough at set times, with standardization and an almost continuous feeding of the machines. As shown in Fig. 3.6, the carousel (www. sancassiano.com) is formed by a number of stations that move automatically (controlled by a Programmable Logic Control, PLC), rotating and occupying different positions at set times. It passes from the first position (raw materials are dosed into the first bowl) to intermediate mixing and kneading positions up to the last position where the dough is discharged and poured out into the feeding hopper of the next machine. The time between the first and the last position corresponds to that necessary to develop a mass with the desired rheology and texture characteristics. In recent years, the companies working in this sector have put forward solutions for further improvement of the automation and versatility of this stage of the industrial process. The different stages of dough development and leavening are controlled by PLC in robotized plants. A variable number of bowls are handled and moved by a robot shuttle which takes each bowl from a parking area and subjects it to the various work stations, on the basis of the sequence of the production cycle. The advantages of this plant are many, including high flexibility, capacity to satisfy all types of technological cycles (direct, indirect, etc.), possibility of feeding several

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lines in parallel (even with different types of dough) and high levels of hygiene and cleaning.

3.4.3.2

Leavening

The production of any leavened baked goods concerns the transformation of the semi-solid mass of the dough, a kind of emulsion with a continuous phase represented by hydrated gluten that surrounds the starch granules, and a dispersed phase, consisting of microbubbles of air, into a foam, where the continuous phase retains considerable volumes of gas (Table 3.2). Leavening is the stage associated with the significant expansion of the original volume of the mass. This is possible thanks to the viscoelastic properties of the dough, and in some cases also to the presence of emulsifiers. The number of alveoli retained in the mass upon completion of the mixing is estimated to be between 102 and 104 per mm3 [50]. Volume expansion can be obtained using biological and chemical leavening agents and also through the physical approach. In this latter case, the inclusion of air follows intense mechanical actions during mixing. Mixed leavening is also considered. Microalveoli incorporated during mixing (physical leavening) are further expanded following the chemical leavening in the oven. Danish pastry, used for making particular sweet products, is obtained from mixed leavening: the dough is first biologically leavened, then formed with a lamination process to distribute the fat in thin and alternate layers within the dough. Although CO2 is considered the major gas responsible for the development of dough volume, other gases and low-boiling substances may interfere with the overall volume of the dough. For instance, ethanol is solubilized in the aqueous stage of the mixture and forms an azeotrope with a boiling point of 78°C and water vapour. The most obvious phenomenon associated with leavening is the volume expansion. The CO2 produced is solubilized firstly in the aqueous stage of the dough. Once saturation is reached, the gas settles in the bubbles entrapped in the dough gradually dilating and expanding them, without any breakages. The pressure inside the alveoli increases but the dough reacts by stretching thanks to gluten viscoelasticity. The high diameter of the bubble makes it possible to balance the overpressure that is created. The film (ca. 1 mm) created by surfactants, soluble proteins, polar lipids, or pentosans, on the surface of the alveolus plays the principal role in this phenomenon [45, 50, 51]. The leavened dough is therefore a foam consisting of a semi-solid aqueous phase where gas bubbles are distributed. The coalescence of these gas bubbles is delayed as long as the lipoprotein film is able to expand, reducing its thickness. Its breakage is associated with the merger with adjacent bubbles. Acidification during sourdough fermentation also influences the rheological properties of the dough. As shown by extensographic analyses, the acidification determines the full maturation of the dough. The extensibility of the dough is modified so that it can better support the dividing and final moulding stages. A fully maturated dough will break clean and sharp with minimum resistance to pull [47]. The reasons for this behaviour are numerous, complex, and only partly understood.

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They are variously attributed to the continuous and progressive hydration of the proteins, to the presence of metabolites (CO2, organic acids) that determine a new organization of gluten, and to changes of the aqueous phases where polymers are immersed. In some cases, the rheological changes are hidden by amylase and protease activities that occur simultaneously during leavening but with opposite effects.

Fermentation and Proofing Rooms At the artisanal level, fermentation is carried out in chambers or cabinets (for limited daily production) where the temperature and humidity are kept constant. The recommended temperature ranges between 27°C and 37°C and the environmental humidity has to be above 75–80% [47]. Lower relative humidity causes the formation of a surface skin that impedes dough development during baking. Optimal temperature and humidity conditions are maintained thanks to air conditioning systems. The baked goods are usually arranged on trays located on mobile trolleys that facilitate movement. Long fermentation times, especially in the sponge-and-dough methods, require one to work overnight. Today, equipment called the “retarder” or “sponge conditioner” allows the optimization of the operations by controlling the kinetic of fermentation at low temperatures [41]. These machines are thermostatic chambers that allow one to control and monitor the temperature (from −10°C to +35°C), through the presence of refrigerator groups and resistors, and the relative humidity. After mixing, the dough is placed inside the chamber where the temperature is lowered and then slowly raised to achieve the fermentation. This optimization of the process reduces or eliminates the night shifts, and distributes the work during the day, guarantying fresh baked goods also in the evening. At the industrial level, the proofing chambers usually consist of a tunnel with dynamic transport and automated controls of the environmental variables. The dough is placed on trays or belts and proceeds towards the exit, thanks to catenaries. The tunnels are sized so that the leavening time coincides with the time needed for the dough to travel inside.

3.4.3.3

Dough Makeup Operations

As briefly described before, the two leavening stages are alternated by dividing, and rounding and moulding operations which were previously carried out manually. Currently, these operations are performed by automated machines with different working capacities. Generally, dividing is carried out within the shortest possible time on a volumetric basis. The fermented dough is inserted into a chamber with a piston whose course is directly proportional to the volume to be divided. The individual piece of dough is cut off by a knife, then ejected from the chamber and shaped. This operation is usually alternated with a period of rest necessary to allow the dough to recover from

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physical stresses associated with compression, followed by cutting and shearing. The working of the divider is completed by rounding and moulding the individual pieces with different machines. Generally, three steps are carried out: the sheeting of dough pieces into a uniform layer, the rolling into a cylinder by means of a rounding or curling machine, and compression to obtain the desired shape.

3.4.3.4

Baking

Baking is considered the most important stage of the entire cycle. During baking, the dough heats up and loses humidity. Heating occurs from the outside towards the inside, water loss occurs in the opposite direction. These two phenomena cause multiple changes, differing in their physical, chemical and biochemical nature and intensity, according to the temperature and the area of the dough. The change from the foam state to the sponge state [52], and the diversification between crust and crumb are observed. The sequence of changes (Table 3.5) is different according to bread area. The temperature inside the dough is always below 100°C, while on the surface it reaches 180–200°C. As soon as the leavened dough is inserted into the oven, the fermentative activity increases [38] until microbial death occurs at temperatures higher than 50°C. Heating causes a further significant volume expansion (oven spring) of about 40% of the volume compared to the leavened dough, corresponding to an increase of the surface area of 10% [49]. The volume occupied by gases, CO2, ethanol vapours, water vapour, increases as the temperature increases. Starting from 70°C, the chemical and biochemical transformations of the macromolecules stabilize the complex. A porous network of interconnected alveoli separated from each other by a solid matrix with very fine walls is formed [52]. During this passage, proteins and starch achieve new properties. The gluten is denatured, completely loses its extensibility and achieves elasticity. The starch swells up and gelatinizes. The intensity of these two phenomena depends on the distance from the geometric centre of the dough. Baking is completed when, even in the most internal part of the dough, the temperature has reached values that promoted the structural consolidation. The temperature at the centre point has to be in the range of 90–95°C to prevent collapse due to a non-rigid structure [52]. Because of the temperature gradient that is created in the dough during baking, the surface, which is exposed to the oven temperature from the beginning, reaches the sponge state much more quickly than the internal part. The surface areas, therefore, become more and more dehydrated and permeable, facilitating evaporation and the release of the water vapour that is generated and accumulated within. Once baking is completed, the crust has a humidity of less than 5% [2]. This value ensures friability and crispiness. The internal crumb retains higher humidity and remains soft and light. The complex chemical reactions that occur during baking are of marked importance for the aroma and taste of baked goods. The starch in the crust is degraded into dextrins between 110 and 140°C. Caramelization starts at 140–150°C and continues, producing pyrodextrins, at higher temperatures. Proteins irreversibly react with

Evaporation of gases Progressive drying Non enzymatic browning Water migration towards crust Structure strengthening

According to bread size

a

Crumb

Crust

Macroscopic level

Crumbling tendency

Loss of typical flavour

Starch gelatinization

Water retention by non-starch polysaccharides Change of fat structure

Appearance of “stale bread” flavour

Increase in hardness

Loss of crispness for increase in crust moisture

Macroscopic level

During storage

Protein/sugar interaction Dextrinization/caramelization Maillard reaction Protein coagulation

Molecular level

Table 3.5 Changes during baking and storage of breada During baking

Water migration exchange at inter and intra macromolecular degree Interaction among aromatic compounds/macromolecules Oxidative phenomena

Starch retrogradation

Water migration from crumb to crust macromolecules

Molecular level

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sugars in the Maillard reaction, forming a number of compounds responsible for the colour and typical aroma of the crust [53]. If the intensity of the Maillard reaction does not exceed certain limits, these effects contribute to the sensory properties. On the contrary, the protein–sugar interactions lead to unavailable lysine and, in advanced stages, cause the synthesis of toxic compounds. The International Maillard Reaction Society site (http://imars.case.edu) provides more information on the nutritional and sensory properties of baked goods. The intensity of the colour of the crust is strictly related to baking temperature, while the thickness of the crust is influenced by baking time. Baked goods undergo a loss of weight during baking, a key step of the process. Usually, bakers aim to obtain the highest yield according to the values of humidity which are allowed by national regulations. In that sense, the practice of including vapour in the oven during the initial baking is justified. The vapour condenses on the surface of the dough, accelerates heat transfer to the dough, slows down evaporation, and decreases weight loss [45]. As a further positive effect, the viscoelastic properties are retained for a longer time on the surface of the dough, allowing a higher development of the baked goods. In the oven, the heat transfer towards the dough occurs by radiation from the walls and by air convection. Inside the dough, the transfer occurs by conduction. Because the temperature is usually between 200 and 230°C, the kinetic of baking is quite slow and provides the consolidation of the internal structure without unacceptable scorching of the crust.

Ovens Regardless of the characteristics of the oven, the baking floor is called the sole or deck and the upper part of the chamber the crown. Ancient ovens, often made of stone or bricks, had a single chamber where combustion and baking occurred. This type of baking is referred to as a “direct firing system” because the combustion gases are in contact with the dough. The initial temperature reaches 350–400°C and decreases during baking to 160–170°C. These ovens, today used only for special traditional or typical breads (e.g. Italian Altamura bread or Arabic bread), were replaced by ovens with an “indirect firing system”, where the combustion area is separated from the baking chamber and the combustion gases do not come in contact with the dough but circulate in tubes above and below the baking surface [47]. This configuration provides a higher uniformity of heating and guarantees hygiene. The heat transfer may be improved using forced air systems inside the chamber (ventilated ovens) or forcing the circulation of the combustion fumes into tubes via a ventilator (Cyclotherm ovens). This method also improves the heat exchange thanks to the repeated passage of the combustion fumes within the tubes positioned above and below the conveyor. Heat is transferred to the

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Fig. 3.7 Modern rack oven (Courtesy of Rational AG, Germany)

dough mainly by radiation. Several oven doors are installed, which change the flow of the fumes towards the upper or bottom area of the chamber to optimize baking based on the dough characteristics. Systems that provide a better heat yield, with an energy saving that may reach 30% compared to those from the conventional thermal cycle ovens, are also available. In artisanal bakeries, the most common ovens have fixed decks with separate chambers which are arranged vertically or consist of a cabinet equipped with a rotating rack carrying trays or frames (Fig. 3.7). In the former ovens, baked goods have to be loaded and unloaded by hand using long peels or special loading devices (Fig. 3.8). This operation requires time and skill. More recent solutions make it possible to vary baking conditions for each chamber. In the rack ovens, baked goods are placed on the pans or trays located on the rack (often during the leavening stage) that is inserted into the oven and is rotated to give a better uniformity during baking. In industrial bakeries, baking is a continuous operation performed in a long horizontal tunnel with different sections or zones, each one having its own burner and where the temperature is variable. Shutters control the evacuation of the water vapour which accumulates in the chamber towards extraction flues. Baking time is determined by the speed of the belt that transports baked goods and by the length of the oven. According to the heating system, ovens are heated by gas, fuel oil or electricity. Microwave ovens combined with traditional ovens are also proposed, but their application is suitable only for specific industrial purposes.

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Fig. 3.8 Artisanal bakery (Courtesy of Wizard Bakery Company, Germany)

3.4.4

The Production Units

3.4.4.1 Artisanal Production The equipment of a modern artisanal baking laboratory is shown in Fig. 3.8. Although it covers a limited surface area, the solution is highly rational as it combines machineries based on the different stages of the technology process. The mixing areas (Fig. 3.8a) are adjacent to the shaping areas (Fig. 3.8b), while the leavening section (Fig. 3.8c, d) is next to the oven area (Fig. 3.8e, f) to facilitate the movement of the dough and its introduction into the oven.

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3.4.4.2

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Industrial Production

At the industrial level, processing is carried out continuously. All the different stages, which are identical to those of the artisanal process, are connected via conveyor belts that carry the semi-finished product to the next stage of the cycle. Rational solutions (see www.wpib.de/; www.itecaspa.com/; www.esmach.it/) provide long horizontal systems with linear transportation to avoid bends or turns. Modern plants consist of completely automated systems of mixing area, controlled by a robotized centre that exclude manual work for dosage of the ingredients, mixing and movement of the bowls to the successive stages. After cutting and shaping, the dough comes to the continuous proofing chamber, whose horizontal or vertical development is proportional to the time needed to complete this stage. Baking under a continuous belt oven is followed by a long cooling stage, which is controlled by circular net transporters.

3.5

Quality Assessment of Dough Baking Properties

Apart from information on the composition of the raw material, a complete knowledge of the changes that occur during the whole technological process is necessary to assess the quality of baked goods. Many instruments and techniques have been developed for this purpose.

3.5.1

Rheology and Descriptive Empirical Measurements

Rheology is the study of the flow and deformation of materials in response to the application of mechanical force. The force is usually defined in terms of stress, the amount of force applied per unit area, with strain being the resulting deformation. The rheological features of the dough are important throughout the bread-making process and determine the quality of baked goods [54]. Rheological measurements are carried out to obtain a quantitative description of the mechanical properties of the materials as well as information related to their molecular structure and composition [54]. Usually, rheological techniques are classified based on the type of strain imposed (e.g. compression, extension, shear, torsion) and on the relative magnitude of the imposed deformation (e.g. small or large deformation). The main techniques used for measuring the properties of cereals are descriptive empirical techniques and fundamental measurements.

3.5.1.1

Quality Assessment of Flour and Descriptive Rheology of Dough

After mixing, subjective manual assessments of the dough were used for a long time to indicate whether it was suitable for processing and baking. Over time, a significant

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use of descriptive empirical measurements of rheological properties was observed. Devices such as the Penetrometer, Texturometer, Consistometer, Amylograph, Farinograph, Mixograph, Extensigraph, Alveograph and Rheofermentometer were developed [55] (Fig. 3.9). Different users may have different requirements. For instance, plant breeders require rapid and automated tests which use small amounts of sample, while millers and bakers require rapid and reliable tests to assay the baking quality [56]. All the tests mimic the complex responses (deformations) in the dough when subjected to various stresses (mixing, overpressure during leavening and baking, etc.). Nevertheless, the behaviour of the dough matrix is non-linear since the deformation is not proportional to the strength that determines it. For instance, the dough appears harder and stiffer when deformed slowly [57]. Therefore, the results from these tests provide useful information only if the nature and intensity of the deformations are similar to those that occur during in situ processing. According to these considerations, the definition is of imitative or empirical or descriptive rheology [45]. Empirical rheology is divided into two main classes [57]. The first class includes instruments that measure the viscosity variation of the dough during mixing/torsion at room temperature (e.g. Brabender Farinograph, Micro-DoughLAB, Mixograph) or according to a gradient of temperature (e.g. Mixolab). The Farinograph (www. brabender.com) measures the water absorption, dough development time, stability and softness. The Mixograph (www.national-mfg.com) determines the mixing time, maximum resistance and tolerance [57]. However, to determine the mixing properties of the dough, mechanical parameters such as mixer speed, mixing bowl capacity, and mixer geometry have to be precisely controlled. The Mixolab (www.chopin.fr) is a relatively new instrument which resembles the Farinograph and Viscoamylograph in terms of performance. It determines in real time the torque (Nm) produced by the dough between the two blades and, once the dough is formed, the device measures its behaviour as a function of time, mixing development and temperature. The second class includes equipment with devices that record and quantify the elastic characteristics of the dough, which are correlated to the resistance that the dough opposes to stretching that is protracted until rupture occurs. The most used are the Alveograph (www.chopin.fr) and the Extensograph (www.brabender.com). The Extensograph performs an extensional test where a cylindrical dough sample is clamped horizontally in a cradle and stretched by a hook which is placed in the middle of the sample and moves downwards until the rupture occurs. The Alveograph uses air pressure to inflate a thin sheet of dough and measures the resistance to expansion and the extensibility of the dough by providing the measurement for maximum over pressure, average abscissa at rupture, index of swelling and deformation energy of dough. Another interesting instrument is the Rheofermentometer (www.chopin.fr). It enables the measurement of gas production and retention, dough permeability, and volume and tolerance during leavening. It is used to determine the quality of the flour, the fermentative activity, the quality of the protein network, and the activities of additives to be selected. Through height and pressure sensors, it measures the fermentation and development of the dough under a weight imposed on it. During recent years, the Texture Analyzer (www.stablemicrosystems.com) has found

3 Technology of Baked Goods Fig. 3.9 Representation of the main tests used for the quality assessment of dough baking properties

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increasing application. The instrument provides information about a long list of textural properties: hardness, brittleness, elasticity, cohesiveness, stickiness, gumminess, springiness, consistency and fracturability. Kieffer et al. [59, 60] developed a micro-method for extension tests. The Kieffer Extensibility Rig is a type of miniature of the Brabender Extensograph and is used in combination with various mechanical testing machines. The cylindrically shaped dough is clamped at both ends and is extended uniaxially by a hook that moves upwards. The Dough Inflation System was introduced in the early 1990s and it was developed based on the concept of the Alveograph. It measures the stress and strain relationships based on the inflation of a sheet of dough through a biaxial extension test and operates at strain rates lower than the Alveograph. The deformations involved in biaxial extension tests are preferred as they are more relevant to the type of deformation of the dough around an expanding gas bubble during proofing and baking. All the above instruments provide a great deal of information on the quality and performance of baked goods. Nevertheless, they are purely descriptive, frequently disruptive, and they depend on the type of instrument used, the size and geometry of the test sample and the specific conditions under which the test is carried out [45, 61].

3.5.1.2

Fundamental Measurements

The study of the effective mechanical properties of dough concerns fundamental rheology. It requires sophisticated equipment and considerable experience. It measures absolute and not relative parameters, providing results that are independent from the particular instrument used. A material is considered solid when it does not change shape continuously when subjected to stress. A material is considered liquid when it changes shape continuously when subjected even to the slightest stress. Dough cannot be classified univocally since it behaves as solid or liquid according to the experimental conditions, and because it shows both solid and liquid features simultaneously. These materials are defined as viscoelastic. The majority of foods show a linear behaviour below a certain deformation value and a non-linear behaviour above. When this limit is exceeded the results depend on the experimental conditions and do not describe the fundamental characteristics of the material [62]. Therefore, the measurements should be carried out during the linear viscoelastic interval of the material. Nevertheless, studies on doughs have also recently focused on non-linear and time-dependent behaviour [63]. Usually, the properties of viscoelastic materials are measured by creep and recovery, stress relaxation or dynamic oscillatory tests. These measurements are usually made on samples placed between two plates of a rheometer. In the creep and recovery test, an instantaneous stress is applied to the sample at rest and the change in strain (creep) is observed over time. When the stress is released, some recovery may be observed as the material attempts to return to its original shape. In the stress-relaxation test, the sample is subjected to an instantaneous strain and the stress required to maintain the deformation is observed as a function of time [62].

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In the oscillatory tests, samples are subjected to deformation or stress which varies harmonically with time. Sinuosoidal and simple shear is typical [62]. This testing procedure is the most common dynamic method for studying the viscoelastic behaviour of food. Results are very sensitive to the chemical composition and physical structure [62]. Using a sinusoidally oscillating deformation of known magnitude and frequency, the phase lag angle between stress and strain is measured and used to calculate the elastic (storage modulus or G¢) and viscous (loss modulus or G″) components of a complex viscosity h*. The rheological behaviour of the dough is determined by protein–protein interactions at large deformations, while starch–starch interactions dominate at small deformations. Therefore, empirical tests correlate well with the results of the baking test [64], as the deformation that occurs is reasonably large compared with the deformation applied during the creep and dynamic rheological tests. In contrast, fundamental tests provide well-defined basic rheological information (viscosity and elasticity) and provide better defined experimental conditions of stress and strain, which allow results to be interpreted in fundamental units. Although various types of tests and instruments have been developed to describe dough performance during processing, it is fair to say that no single technique could completely describe its rheological behaviour.

3.5.2

Innovative Approaches

3.5.2.1

Image Analysis

Recently, image analysis has been introduced to evaluate the quality of foods, including baked goods. This technique uses protocols based on image digitalization at the macro- and micro-structural level through different systems (e.g. scanners, video cameras and microscopes). Image analysis provides a rapid and objective definition of the morphological and densitometrical characteristics of single objects or complex structures (Fig. 3.10). It makes it possible to study and model the phenomena that occur during processing continuously or even on-line [65, 66]. The analysis of an image requires a number of passages: (1) image acquisition in a digital format (a pixel image); (2) image pre-processing, to improve the image while maintaining its original dimensions; (3) image segmentation, to divide the digital image into separate, non-overlapping areas (e.g. to better distinguish the objects from the rest of the image, such as the alveoli in a slice of bread); (4) measurement of objects, to determine their different characteristics (size, shape, colour, texture); and (5) classification, to identify the objects by classifying them into different classes [67]. When used for baked good processing, image analysis allows the determination of several parameters such as the increase of volume, changes of shape, time needed to complete the dough development, extent and distribution of the alveolar structure during leavening, initial increase and successive contraction of volume, and gelatinization of the starch and surface browning during baking [68].

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Fig. 3.10 The use of the image analysis approach in the evaluation of the leavening phase of dough

3.5.2.2

Microscopy

The macroscopic behaviour of the dough depends on its microstructure. The latter is affected by composition, spatial arrangements of the components and types of bond existing [69]. Microscopy is often used to determine the optimal mixing time of the dough, the extent of the development of the gluten and the nature of the gluten matrix [70–72]. Many details are explored via electronic microscopy, both by transmission (TEM, transmission electron microscopy) and scanning (SEM, scanning electron microscopy). To minimize the influence of sample preparation, atomic force microscopy (AFM) may be used. This technique provides high-resolution images of the surface of the starch granules [73]. Confocal laser scanning microscopy (CLSM) has recently found application in the analysis of foods. It offers the possibility of optically dissecting the material and reconstructing the 3D image [74], and to observe dynamic processes such as the formation and growth of air bubbles in the dough during leavening and baking [75]. The different components of the dough may also be simultaneously identified and located, using specific fluorescent markers. Electronic microscopy was also used to study the rupture of the gluten network following freezing and thawing [76]. A very useful instrument for the observation of frozen matrices is the cryo-SEM. This instrument shows the ultrastructure of the starch–protein associations and the state of the gluten fibrils forming the protein network [77].

3.5.2.3

Spectroscopy

The request for quick, reliable and easy to use methods that provide automation or on-line applications is increasingly frequent. Near-infrared spectroscopy (NIR) satisfies these requirements. This technique is based on the acquisition of information from a sample via the interactions that occur between its molecules and the electromagnetic waves in the near infrared. NIR offers the possibility of analyzing the matrix in a non-destructive way, does not require the use of reagents, and is highly informative. NIR spectra allow the simultaneous quantification of the various components or information regarding the mutual relations between them [78]. Recently, the NIR technique was used as a potential on-line sensor to monitor

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processing [79, 80]. To completely exploit the potential of NIR, advanced chemometric techniques are needed for the interpretation of spectral data which are arranged in wide bands with overlapping peaks that originate from the different components present in the matrix [78]. NIR spectroscopy is largely used to quickly determine the chemical composition of caryopses and flours. Other studies reported its application to determine the technological quality of the flours [81–84], to evaluate the molecular interactions between the dough components (water-protein-starch) [85, 86], and to monitor mixing [87], leavening, and staling [78]. Recent developments concerned the acquisition of information on dough via interactions that occur between the molecules and the electromagnetic waves in the infrared medium (MIR, mid-infrared spectroscopy). Nuclear magnetic resonance (NMR) was also used for baked good processing. It was applied to monitor the distribution and mobility of the water, to investigate the structure of the product and track the staling phenomenon [88–90].

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www.bakerperkinsgroup.com www.brabender.com www.buhlergroup.com www.chopin.fr www.esmach.it www.fao.org/docrep (FAO - Food Outlook) www.imars.case.edu www.itecaspa.com www.national-mfg.com www.ocrim.it www.sancassiano.com www.satake-group.com www.stablemicrosystems.com www.wpib.de

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Chapter 4

Technology of Sourdough Fermentation and Sourdough Applications Aldo Corsetti

4.1

Definition of Sourdough

Sourdough technology is widely used in bread making and cake production as it confers distinctive characteristics, high sensory properties and shelf life to the resulting products (Table 1). Sourdough is “a mixture of wheat and/or rye flour and water, possibly with added salt, fermented by spontaneous (from flour and environment) lactic acid bacteria and yeasts which determine its acidifying and leavening capability. These activities are obtained and optimized through consecutive refreshments (or re-buildings, replenishments, backslopping)” (3–5). The term refreshment deals with the technique by which a dough made of flour, water and possibly other ingredients ferments spontaneously for a certain time (possibly at a defined temperature) and it is subsequently added as an inoculum to start the fermentation of a new mixture of flour and water (and possibly other ingredients). When applied for a defined interval of time such a process provides a sourdough with constant and repeatable leavening and acidifying performances reliant on the growth of lactic acid bacteria and yeasts that are well adapted to the environment. After the preparation of the sourdough, the refreshment technique is aimed at maintaining the metabolic activity of the microbial communities at all times (6). When the sourdough is added to a mixture of water and flour to start consecutive propagations (refreshments) to obtain the final mass or full sour to be used as the leavening agent, it can be designated the “mother sponge” (2, 4). Generally, a sourdough contains a variable number of lactic acid bacteria and yeasts, ranging from 107 to 109 cfu/g and 105 to 107 cfu/g, respectively, with a ratio of about 100:1 (7).

A. Corsetti (*) Department of Food Science, University of Teramo, Teramo, Italy e-mail: [email protected] M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, DOI 10.1007/978-1-4614-5425-0_4, © Springer Science+Business Media New York 2013

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Table 4.1 Characteristics of sourdough bread versus baker’s yeast bread (adapted from (1, 2)) Characteristics Sourdough bread Baker’s yeast bread pH 3.8–4.6 5.3–5.8 Lactic acid 0.4–0.8% 0.005–0.04% Acetic acid 0.10–0.40% 0.005–0.04% Bread volume 0.22–0.30 £0.20 Flavour Complex aroma and flavour Staling Slow Rapid Shelf life Good protection against microbial High sensibility to bacteria and contaminations mould spoilage Some nutritional Optimal phytase activity and Low phytase activity, decalcifying aspects hydrolysis of phytic acid effect responsible for ion (Ca2+, Fe2+, Mg2+ etc.) binding Free amino acid concentration Free amino acid concentration increase similar to that of flour Decrease of glycemic index

The main role of lactic acid bacteria (mainly obligately and facultatively heterofermentative lactobacilli) is in the acidification process while yeasts mainly account for the leavening of the dough by releasing CO2 (4).

4.2

Sourdough Preparation and Storage

Sourdough (mother sponge) preparation can be fulfilled through many different protocols. The main objective is to obtain a leavening agent that contains welladapted resident microorganisms. Such microorganisms have to produce sufficient CO2 to leaven the dough, and organic acids and other metabolites to provide rye or wheat bread with good texture and sensory properties, and extended shelf life (3). Two classical procedures, for example the French and American systems, are discussed below.

4.2.1

The French System

Mother sponge preparation for obtaining the French “pain au levain” begins with a quite firm wheat flour dough (dough yield, DY of 150–152, see Sect. 4.6.2) with addition of salt and malt. This dough undergoes a first fermentation step lasting ca. 24 h. This corresponds to the early fermentative activity of flourresident yeast and lactic acid bacteria, which results in a low CO2 and organic acid release (2). The decrease of pH induces the activity of flour endogenous proteases which together with bacterial hydrolytic enzymes act on gluten and

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lead to a lower dough firmness. The second step begins with the first refreshment which is aimed at introducing oxygen and new fermentable carbohydrates into the mixture to stimulate microbial growth and activity. The refreshment is obtained by adding a quantity of flour corresponding to the weight of the previous fermented dough and a quantity of water to bring the DY to a value (e.g. 148) lower than the previous one. This dough ferments quickly and represents the starting dough for the next refreshment. By applying such a procedure a sourdough with a steady fermentative and leavening capability is obtained. During the last step, each refreshment is carried out at a regular interval of time (e.g. 7–8 h), with the aim of maintaining an equilibrium in the ratio between microbial communities (2). According to Calvel (2), when the dough volume increases by three to fourfold with respect to the initial dough, a new refreshment should be performed. The mother sponge (levain chef), which is obtained following the above procedure, represents the dough used to prepare the full sour needed to leaven the bread-dough.

4.2.2

The American System

The American system relies for the mother sponge preparation on a mix of water and wheat and/or rye flours. In contrast to the French system, the value of DY ranges from 225 (liquid dough with a ratio water:flour of 1.25:1) to 250 (liquid dough with a ratio water:flour of 1.5:1). These values of DY remain unaltered throughout the consecutive refreshments (8). The time and temperature of fermentation are strictly controlled during each phase. In the first step, the waterflour mixture ferments at 32–35°C for 24 h in order to acidify the dough. At the end of this step, the first refreshment is obtained by adding flour and water to the previous fermented dough, without changing the value of DY. After 8 h of fermentation at 32–35°C, the second refreshment is carried out and the dough ferments for another 16 h. After the above procedure is applied, refreshments are carried out every 8–16 h between which the dough is allowed to ferment at 24–27°C. With the aim of calculating the amount of water and flour to be added at each replenishment stage a multiplicative factor of 4 is considered. Under these conditions, the weight of the fermented dough is multiplied by 4 every two refreshments. This allows one to determine the quantity of water and flour to be used for the next refreshment and the ratio between the two ingredients is maintained to 1.25:1. By using this system the value of DY remains constant until a sourdough with a pH value of 3.6–3.8 and TTA (total titratable acidity) of 16–20 ml NaOH/20 g of dough (as calculated following the American system) is obtained. A last fermentation of 8 h at 24–27°C is needed to confer to the sourdough the sensory and leavening performances of the mother sponge. Generally, a sourdough with the above characteristics is obtained in approximately 5 days, after which it is maintained in an active state by storage at low temperature (e.g. 4°C) and subjected to refreshment at least once a day (8).

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A. Corsetti

Sourdough Storage

The liquid sourdough (DY 225–250) that is frequently used by bread manufacturers in the United States is stored at 1–2°C, after rapid refrigeration, or at 4–5°C. It is used to start a new fermentation within 2–3 days, without the refreshment step. In the case of prolonged storage (10 days) one or two refreshments are needed to activate the metabolism of the lactic acid bacteria and yeasts. A prolonged storage of the sourdough for a few months at 4–5°C is possible when the ratio between water and flour is reduced by adding flour at ca. 0.43:1 (30% water: 70% flour). In this case, a firm sourdough, with DY of 143, is produced. Such a storage type necessarily requires sourdough reactivation (at least two refreshments) before use (8). In many artisan bread preparations, different and often empirical storage techniques are applied. Generally, as a consequence of the daily schedule of bread manufacture, a portion of the sourdough is refreshed at least one time before its use. Nevertheless, by applying a separate storage protocol, part of the mother sponge can be stored at low temperature (4–6°C) for some weeks after putting it in a cloth bag tied with string (9). Refreshments are needed before reusing such a sourdough in a bread-making protocol. In some cases sourdough can be frozen and reused after refreshment.

4.3

Classification of Sourdoughs

Sourdough bread making is an ancient biotechnological process and various protocols for its use are applied in many countries. On the basis of the technology applied, sourdoughs have been grouped into three types (10), to which a fourth type, named sponge-dough, can be added.

4.3.1

Type I Sourdough

Traditional sourdoughs whose microorganisms are kept metabolically active through daily refreshments are included in this group. Type I sourdoughs are generally suitable for achieving dough leavening without addition of baker’s yeast; the dough propagation described above for French and U.S. sourdoughs are examples of Type I sourdoughs. Generally, a three-stage protocol is applied relying on three refreshments over 24 h in order to obtain the leavened dough to bake. Each step is characterized by a given DY as well as fermentation temperature and time. At the end of the last step of fermentation the sourdough is used as the leavening agent; thus it can be considered as a natural starter culture containing many microbial strains (11). In wheat and/or rye flour sourdoughs, dominating strains belong to the species Lactobacillus sanfranciscensis which can co-exist with other obligately

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heterofermentative lactic acid bacteria such as L. pontis, L. brevis, L. fermentum, L. fructivorans and with the yeasts Candida milleri, C. holmii, Saccharomyces cerevisiae and S. exiguus (recently renamed Kazachstania exigua).

4.3.2

Type II Sourdough

Sourdoughs obtained through a unique fermentation step of 15–20 h followed by storage for many days belong to this group. Type II sourdoughs are generally not suitable for achieving dough leavening but are used for dough acidification, and as dough improvers. These sourdoughs are generally liquid (DY of ca. 200) and they are produced at the industrial level using bioreactors or tanks at a controlled temperature that exceeds 30°C. Such a protocol aims at shortening the fermentation process (12). During storage, a portion of the mature sourdough can be used as the inoculum with the aim of acidifying the dough and enriching it with aroma and flavour compounds which are characteristics of sourdough baked goods. On the basis of the long fermentation time, high DY, and temperature of fermentation, lactobacilli such as L. panis, L. reuteri, L. johnsonii, and L. pontis, which are resistant to low pH, dominate these sourdoughs (10, 13). Spontaneous flour yeasts are inhibited and, consequently, the leavening of the final dough is obtained by adding commercial baker’s yeast. Through a combined approach consisting of culture-dependent and culture-independent systems, Meroth et al. (14, 15), by using an experimental model based on a laboratory scale-fermentation, showed a different prevalence of lactic acid bacteria and yeasts in type I and type II rye-based sourdough, started with a mixture of commercially available sourdough starters and baker’s yeast. In particular, L. sanfranciscensis, L. mindensis and C. humilis dominated the type I sourdough. L. crispatus, L. pontis, L. fermentum and S. cerevisiae prevailed in the type II sourdough propagated at 30°C; the same sourdough propagated at 40°C showed a dominance of L. crispatus, L. panis and L. frumenti as well as the disappearance of all the yeasts that had been added with the starter mixture. Finally, L. johnsonii, L. reuteri and C. krusei characterized a type II sourdough fermented at high temperature (40°C), but produced with rye bran instead of rye flour.

4.3.3

Type III Sourdough

Type II liquid sourdoughs, which are dried/stabilized after preparation, are named type III sourdoughs (12). They are mainly used at the industrial level as their quality is more constant compared to type I sourdough, and they are simpler to manage and less time consuming. On the basis of the preparation method, type III sourdoughs are dominated by drying-resistant lactic acid bacteria such as Pediococcus pentosaceus, L. plantarum and L. brevis (10). A detailed description of this type of sourdough and its industrial application are reported in Sect. 4.8.

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Sponge Dough

The sponge dough is aimed at acclimatizing baker’s yeast (S. cerevisiae) and improving swelling of the flour components, loaf volume, taste and flavour of the bread and its shelf life. It is an indirect process which, from a technological and microbiological point of view, could be considered as an intermediate procedure between straight-dough (or a direct process in which just baker’s yeast is used to start the fermentation) and sourdough (an indirect process in which fermentation starts without the addition of baker’s yeast). Sponge dough is obtained in two steps: in the first dough (pre-dough) the baker’s yeast is mixed with a part of the flour and water of the recipe, while the second dough is obtained by adding the rest of the flour, water and possibly other ingredients to the fully fermented pre-dough. Depending on the type of bread, the length of pre-dough fermentation can vary from 3 to 20 h and as a consequence, various percentages of flour and yeast, besides different combinations of DY and temperature can be applied in this step (16, 17). As in the case of longer fermentations lactic acid bacteria present as contaminants from either baker’s yeast or flour grow in the dough reaching typically more than 108 cfu/g and contribute to the overall quality of baked goods (4), the sponge dough can be included in the category of sourdoughs.

4.4

Examples of Sourdough Applications

Once produced, the sourdough is generally submitted to various refreshments either to activate the microbial metabolism or to increase the dough mass (Fig. 4.1). Different types of sourdough (e.g. type I, II or III) are used for the manufacture of various leavened baked products, which range from traditional Italian or French wheat bread, white pan bread, rye bread, San Francisco bread to traditional cakes famous throughout the world such as Panettone. Examples of those sourdough applications are given below.

4.4.1

A Traditional Italian Sourdough Bread: The Altamura Bread

The Altamura bread is the first European bread that received the PDO (Protected Denomination of Origin) status. It is manufactured in the Apulia region (Italy) using specific cultivars of durum wheat (Triticum durum) flour and the technology is based on type I sourdough. The full sour is obtained by a three-stage procedure in order to gradually increase the amount of leavened dough. At each step, water and durum wheat flour are mixed with a previously fermented dough, which is added at the proportion of ca. 20% based on flour weight. On the basis of the ratio between

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Water

1st refreshment – fermentation at 25°C for 5 - 6 h

Flour

Water

2nd refreshment – fermentation at 28°C for 7 - 8 h

Flour

Water

3rd refreshment – fermentation at 25°C for 2 - 3 h

Flour

Full sour

Mother sponge stored till using for the next bread-making

Leavening agent for bread production

Fig. 4.1 Representative flow sheet to obtain a full sour from a mother sponge using a three-step procedure. Fermentation time and temperature given in the example can change depending on bread-making protocol (Adapted from (1))

the ingredients that are indicated in the original recipe (100 kg of durum wheat flour + 20 kg of sourdough + 2 kg NaCl + 60 l of water) and assuming a full sour with a DY of 160, which contains 12.5 kg of flour, the final dough should have a DY of 162. As in many traditional productions, the time and temperature used at each stage are not defined but variously determined based on the bread-makers experience.

4.4.2

French Bread (Pain au levain)

Although various protocols based on one or two steps are used to obtain an adequate quantity of full sour (levain tout point), a three-stage system is usually applied to prepare the traditional wheat flour sourdough French bread, according to the type I

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sourdough procedure. The first step consists of the mixing of a part of the mother sponge (levain chef) with flour and water to achieve around four times the initial mass. This dough (DY 160) ferments for 1.5–2 h at ca. 25°C and is named levain de première (fresh sour). It is used as a starter to obtain a dough with a DY of ca. 185, which ferments for 7–8 h at a temperature slightly higher than the previous one. Those parameters, including the long mixing time to oxygenate the dough, stimulate the growth of yeasts and the leavening capability of the levain de seconde (basic sour). By using this dough as the starter for the third refreshment, the last dough or levain tout point (full sour) is obtained after a short fermentation time (ca. 2 h) with the aim of controlling the hydrolytic activities of the dough and of preserving the gas-retaining and bread-making capabilities. The levain tout point is used at the proportion of 25% with respect to the mass of the final dough and the dough is ready for baking after 30 min of leavening (2).

4.4.3

Rye Sourdough Bread

As reported for French bread, the rye or wheat/rye flour sourdough bread is obtained using single or multi-step protocols, depending on the type of bread making and product. The “three-stage sourdough process, basic-sour overnight” requires ca. 15–20 h and, as described by Spicher and Pomeranz (17), represents a good reference model for type I sourdough application in rye-flour-based bread making. The first stage leads to the anfrischsauer (fresh sour), which, in turn, is started by the anstellgut (mother sponge), a commercial starter or a part (ca. 10–15% of the total dough weight) of a full sour (vollsauer) from a previous bread making. The anfrischsauer has a DY of 200–250 and ferments for ca. 6 h at 25–26°C. Afterwards, it contains a high number of yeasts and it is used to start a new dough based on rye flour and water (DY of about 160–180). After 5–8 h of overnight fermentation at 26–30°C, lactic acid bacteria grow and the grundsauer (basic sour) is obtained. A further refreshment (3 h at 30–33°C), using the basic sour as the starter for a mix of rye flour and water (DY 180–200), leads to the vollsauer (full sour), which has values of pH and TTA of 4.0 and 10.5 ml NaOH 0.1 N/10 g of dough, respectively. A part (40%) of this sourdough represents the natural starter for the final dough (DY 170), which contains rye flour and an equivalent amount of wheat flour in the case of a rye/wheat flour bread preparation. The dough is baked after dough resting of 10 min at 28°C, followed by proofing.

4.4.4

White Pan Bread

The white pan bread is one of the most diffused breads in the United States and represents a good example of bread that is manufactured using type II sourdough. As reported by Kulp (8), the manufacture of this bread relies on a “one-stage system”

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where just one refreshment is needed to mix the liquid full sour with the other ingredients of the recipe. The quite soft dough (DY 175) ferments in two separate steps: the first at room temperature (ca. 25–26°C) for 25–30 min and the second at 35°C for ca. 75–100 min. Before baking, the dough shows values of pH of 4.3–4.4 and values of TTA of 9–11 ml NaOH 0.1 N/20 g of dough. Overall, the liquid starter (full sour) represents 37% of the final dough but different percentages can be used, which depend on the recipe and industrial or artisan levels. In the last case, a lower quantity of starter is used, which requires a longer fermentation period and different working schedule. Because of the high acidity of the sourdough starter, a short mixing time is required due to the increased solubility of the gluten proteins, which decreases the time for development of the gluten network.

4.4.5

Panettone Cake

Panettone is a traditional Italian cake consumed over Christmas and famous throughout the world. Panettone has a soft structure, with regular holes, and characteristic flavour, which is derived from dough ingredients (water, flour, butter, sugar, eggs, salt, and others) and processing. Both at artisan and industrial levels, the sourdough biotechnology is traditionally based on many refreshments (e.g. type I sourdough) and an increased concentration of sugar is added during the last steps (9, 12). Comparable to most other type I sourdoughs, sourdough microbiota are predominantly composed of L. sanfranciscensis. During traditional manufacture, a sourdough with a low fermentation quotient (FQ, see Sect. 4.6.3) is used and time and temperature of fermentation are strictly controlled. In particular, the dough temperature does not exceed 30°C and the temperature of water and other ingredients has to be not lower than 20–22°C. Various studies have attributed the prolonged softness and shelf life of Panettone cake (it is manufactured several months before consumption) to the presence of dextran producing Leuconostoc mesenteroides in the sourdough, which uses sucrose as an exo-polysaccharide (EPS) precursor (12). On the basis of such a finding a new protocol relying on type III sourdough has recently been proposed as an alternative to the traditional manufacture (see Sect. 4.8). A sourdough containing 25% (on dry matter) of dextran, which is stabilized by refrigeration, pasteurization or drying, has been used to shorten the time to obtaining a product with similar characteristics to the traditional Panettone cake (18).

4.4.6

San Francisco Bread

San Francisco bread is traditionally manufactured using the very famous San Francisco sourdough, which possesses a typical microbial community, mainly consisting of L. sanfranciscensis and S. exiguus (renamed to K. exigua). Traditional bread making relies on the type I sourdough. Various refreshments and long

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fermentation times at low temperature are used to increase the concentration of acetic acid synthesized by the obligately heterofermentative L. sanfranciscensis. Recently, a liquid San Francisco sourdough, which is stabilized through pasteurization, has been introduced to the market. Thus, also the type III sourdough is used as an acidifying and flavouring agent for the manufacture of the San Francisco bread in ca. 3 h (no-time-dough process) (see Sect. 4.7) (12).

4.5

Stability of the Sourdough Microbiota During Propagation and Use

In order to keep microorganisms active, type I sourdoughs are, generally, propagated daily. Thus, problems of microbiota stability during propagation and in the use of such a type of “biological starter” arise. In general, it is recognized that in a sourdough ready to be used (e.g. the mother sponge—see Sect. 4.2) for bread and other baked goods production the following basic characteristics occur: – The prevalence of a microbial community different from that of the flour used as raw material (3, 19, 20); – A stable ratio among lactic acid bacteria and yeasts (to the order of 100:1 or higher) (7); – A predominance of facultatively and, especially, obligately heterofermentative lactobacilli over other lactic acid bacteria (3). As reported by Vrancken et al. (21), laboratory sourdoughs based on wheat, rye, or spelt that are backslopped daily, reach equilibrium through a three-step process: (1) prevalence of sourdough-atypical lactic acid bacteria, (2) prevalence of sourdough-typical lactic acid bacteria, and (3) prevalence of highly adapted sourdough-typical lactic acid bacteria (21). In a recent paper dealing with the comparative analysis of seven mature sourdoughs of type I backslopped for 80 days at artisan and laboratory levels under constant technological parameters, Minervini et al. (22) showed that, while the cell density of presumptive lactic acid bacteria and related biochemical features were not affected by the environment of propagation, all sourdoughs harboured a certain number of species and strains, which were dominant throughout time and, in several cases, varied depending on the environment of propagation (21). Moreover, besides stable species and strains, other lactic acid bacteria temporarily contaminated the sourdoughs with differences between artisan and laboratory levels. Interestingly, this occasional succession of strains and species only slightly affected the kinetic of sourdough acidification; nevertheless, it could influence the sensory properties of the resulting leavened baked product. Overall, as a stable microbiota during sourdough propagation and use is essential in order to obtain standard and repeatable final products, the problem of microbial stability in terms of species and

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Fig. 4.2 Persistence of strains of Lactobacillus plantarum (DB200, --♦--; 12H1, --l--; 2MF8, --£--; G10C3, --D--) and Lactobacillus sanfranciscensis (LS6, -£-; LS41, –n–; LS48, —◊—; LS3, —X—) after sourdough propagation at 30°C for 6 h during ten subsequent days (Adapted from (24) and from (23))

strain composition in type I sourdough propagated by applying different endogenous (e.g. type of flour, quantity of water) and exogenous (e.g. temperature/time of fermentation) parameters should be carefully addressed, both in terms of identification and typing of dominant and sub-dominant microorganisms (see Sect. 4.6.1) as well as to select robust, well-adapting and competitive starter strains (23). The robustness of sourdough lactobacilli varies depending on the species and on the strains. While the majority of L. sanfranciscensis strains showed quite a low robustness during daily backslopping performed at the laboratory level (24), selected strains of L. plantarum seemed to share several phenotypic traits that determined the capacity to outcompete the contaminating lactic acid bacterium biota (23) (Fig. 4.2).

4.6

Methods to Evaluate the Performance of the Sourdough

Both microbiological and physico-chemical parameters are used to evaluate the performance of a sourdough. The level of complexity is different and depends on the purpose of the analyses. The microbiological aspect essentially deals with the assessment of the community of lactic acid bacteria and yeasts, as those microorganisms are dominant in a good quality sourdough and are generally present at the ratio of approx. 100:1 (7). Nevertheless, an array of both phenotypic and genotypic methods is necessary to identify the species/strain composition of the dominant and sub-dominant microbiota of the sourdough. An overview of those systems is given below.

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Determination of the Number of Lactic Acid Bacteria and Yeasts

The standard plate count is used to estimate the cell density of both lactic acid bacteria and yeasts. Suitable culture media to assess the number of sourdough lactic acid bacteria include SDB (Sour Dough Bacteria) medium, as originally described by Kline and Sugihara (25); or MRS (de Man, Rogosa, Sharpe) medium (51), with modifications concerning the pH and the nutrient composition, particularly to include maltose and fructose as carbon sources and electron acceptors, respectively, and cysteine as reducing agent (26–28); Homohiochii medium, especially recommended to enumerate obligately heterofermentative lactic acid bacteria (29); SFM (San Francisco medium), containing wheat or rye bran especially useful for lactic acid bacteria as well as lactobacilli (30). To all the above media cycloheximide (100 ppm) can be added to inhibit the growth of yeast when requested. Yeast are generally enumerated on WL medium (31); YPDA medium (32, 33); Sabouraud dextrose agar (34, 35); YGC agar (14); and Malt extract agar (36). To inhibit bacterial growth, all the above media generally contain 50–100 ppm of chloramphenicol.

4.6.2

Phenotypic and Genetic Analyses to Identify and Type Lactic Acid Bacteria and Yeasts

Sourdough lactic acid bacteria and yeasts are generally identified using a polyphasic approach, combining both phenotypic and genotypic assays. Besides morphological and physiological tests, specific biochemical assays are available to identify at species level Weissella spp., Pediococcus spp. and Enterococcus spp. as well as the predominant sourdough lactic acid bacteria, which are often members of the genus Lactobacillus (19). In that case, the miniaturized commercial test (e.g. API 50 CHL, Biomerieux, France) or automated assay (e.g. Biolog system) are frequently applied after a preliminary evaluation based on cell morphology, Gram stain, catalase test, growth at 15°C and 45°C, CO2 production from glucose, and NH3 release from arginine (37). Nevertheless, the high biochemical diversity in the above-mentioned genus, means that the identification scheme must include some chemotaxonomic analysis (e.g. SDSPAGE, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis of total or cell-wall associated proteins) (38, 39) or, mainly, genotypic tests such as 16S rRNA gene sequencing (30). Moreover, on the basis of specifically designed primers targeting species-specific sequences, many rapid identification systems relying on PCR (e.g. multiplex-PCR, Intergenic Spacer Region-based PCR) have been recently applied to sourdough lactic acid bacteria identification (27, 40, 41). Among genotypic systems, PCR-RAPD (Random Amplified Polymorphic DNA) as well as RFLP (Restriction Fragment Length Polymorphism) or PFGE (Pulsed Field Gel Electrophoresis) have been successfully applied for the evaluation of intra-specific differences (e.g. strain typing) (38, 42, 43).

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On the basis of a similar identification scheme as described above, sourdough yeasts can be preliminarily identified by a phenotypic approach consisting of cell and colony morphology, presence and number of spores, carbohydrates, organic acids, alcohols and nitrite fermentation/assimilation tests using commercial kits (e.g. ID32C, API 20C AUX, RapID Yeast Plus System, Rapid Yeast Plus), growth at different temperatures and salt concentrations (44). As for lactic acid bacteria, chemotaxonomy based on fatty acids, proteins and polysaccharides can be successfully applied to yeast identification. The genotypic approach is often based on the sequencing of various ribosomal genes (e.g. 18S, 5,8S, 26S, 5S, and spacer fragments) (45), as well as on the PCR-RFLP of the rDNA 5,8S-ITS (Internal Transcribed Spacers) (34). As for lactic acid bacteria, yeast strain typing mainly relies on the PCR-RAPD technique. Both lactic acid bacteria and yeasts can be identified and monitored during sourdough fermentation by culture-independent systems, for example by PCR-DGGE (Denaturing Gradient Gel Electrophoresis), generally directed toward the amplification of specific variable regions of 16S or 26S rRNA genes of lactic acid bacteria and yeasts, respectively. A similar approach was applied by Meroth et al. (14, 15) to evaluate the effect of type I and type II sourdough fermentation on microbial population dynamics.

4.6.3

Determination of the Physico-Chemical Parameters

The balance between the communities of lactic acid bacteria and yeasts and the composition of the microbial communities in terms of species and strains markedly influences the sourdough performances and the overall quality of related leavened baked goods. The microbial community is influenced by some sourdough characteristics and, in turn, it modifies the chemical composition and physical parameters of the sourdough, which is reflected in the overall quality of the products (Fig. 4.3). An overview of the physico-chemical characteristics of the sourdough, which are related to its performance, is determined through the evaluation of the following parameters: – Dough Yield (DY) – Dough Acidity (pH and Total Titratable Acidity, TTA) – Fermentation Quotient (FQ). Dough Yield (DY). The ratio between water and flour in the dough is indicated as dough yield and it deals with the dough consistency. Considering that different flours have different capabilities to absorb water, doughs of various consistency are obtained having the same DY. DY is calculated as follows: DY = (flour weight + water weight) × 100/flour weight. To consider other ingredients in the formula, the expression is modified as follows: DY = dough weight × 100/flour weight. Overall, a firm wheat flour sourdough has a DY of 150–160. A liquid sourdough shows values close to 200. Intermediate values of DY indicate soft dough (12). It has been observed that both DY and temperature of fermentation markedly influence

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Endogenous factors Carbohydrates Nitrogen sources Minerals Lipids, free fatty acids Enzymatic activities (e.g. proteases, amylases)

Lactic acid bacteria

Raw materials/ Environment Microbiota

composition, ratio, performances Inoculum

Yeasts

Product quality flavour texture shelf-life nutritional value

Contaminating microbiota

Exogenous factors Temperature Dough Yield (aw) Oxygen (redox potential) Fermentation time Number of refreshments

Fig. 4.3 Endogenous and exogenous factors impacting on sourdough characteristics and performances as well as on the overall quality of the final product (Adapted from (4))

the aroma of the sourdough and, especially, the molar ratio between lactic and acetic acids (FQ). Overall, more acetic acid is present in firm dough fermented at 25–30°C, while more lactic acid is found in soft dough fermented at 35–37°C (12, 46). Obviously, on the basis of microbial adaptation to the various environmental factors, the combination of DY and temperature during refreshments markedly influences sourdough microbiota and its performance. Acetic acid plays an important role by influencing many bread properties (flavour, rope inhibition, shelf life), and its content in sourdough can be increased by fructose addition or by aeration in the presence of heterofermentative lactobacilli (47). In many cases, the effect of temperature on the acetic acid content of type I sourdoughs (generally propagated at a temperature of about 25°C), can strictly depend on the yeast activity: at low temperature, yeasts grow and hydrolyze kestose and other fructo-oligosaccharides, with the fructose being available as electron acceptors for a higher production of acetic acid by L. sanfranciscensis via the acetate-kinase pathway (47). At a high temperature (more than 32°C), growth of yeasts is inhibited, fructo-oligosaccharides remain un-hydrolyzed, and L. sanfranciscensis has less fructose available for acetate production (48). pH. The final pH, which ranges from 3.5 to 4.3, is usually considered as an index of a well-developed sourdough fermentation (50). The pH of the sourdough influences the values of pH of the final dough and bread, depending on the amount of full sour that is used as the inoculum. In the case of a standard inoculum of 20% (referred to the dough weight), values of pH that range from 4.7 to 5.4 are usually found in the final dough (50). TTA (Total Titratable Acidity). The value of TTA is a measure of the total organic acids synthesized during sourdough fermentation. TTA is expressed as ml of NaOH

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0.1 N/10 g of dough. The values of TTA range from 30 to 150 ml NaOH 0.1 N/10 g for liquid to 40–220 NaOH 0.1 N/10 g for dried sourdoughs. Nevertheless, the optimal value of TTA for the sourdough depends on the type of bread. Overall, sourdough with high TTA are preferred for bread making with rye flour (48). Fermentation Quotient (FQ). FQ indicates the molar ratio between lactic and acetic acids during sourdough fermentation. After the determination of the concentration of lactic and acetic acids by enzymatic or chromatographic methods, the FQ is calculated as follows: FQ = (g of lactic acid in 100 g of dough/molecular weight of lactic acid): (g of acetic acid in 100 g of dough/molecular weight of acetic acid). This parameter is strictly related to the type of lactic acid bacteria dominating the fermentation and markedly varies depending on the balance between homoand hetero-fermentative lactobacilli. In turn, this balance depends on exogenous and endogenous factors which prevail during fermentation (e.g. fermentable sugar and oxygen concentration, DY, time and temperature, etc.).

4.7 The Use of Stabilized Sourdoughs Versus Active Sourdoughs On the basis of the research progress in the field of bread-making biotechnology, novel types of sourdough have recently been developed with the main aim of improving the flavour of leavened baked products without using traditional protocols of manufacture based on active sourdough. Currently, various commercial preparations of stabilized sourdough are available on the market. They are used for making traditional breads and are based on a mix of tailor-made aroma compounds. Different types of stabilized sourdough are obtained starting from an accurate selection and mix of raw materials (e.g. flour from T. aestivum or T. durum wheat, rye or other cereals) and microbial strains. Dried sourdough is a type of stabilized product useful for this purpose. Different drying protocols can be applied, for example freeze-drying, spray granulation, fluidized bed drying, spray drying and drum drying, among which the latter two can be cited here as the most common for type III sourdough production (12, 48). In both cases, the higher the DY of the starting type II sourdough the higher the resulting TTA value of the derived type III sourdough, which also increases due to water evaporation. In the spray-drying process, the liquid sourdough is dried by using a warm air flux that removes water until the humidity becomes less than 10%. In the drum-drying system, the vapour of the warmed drums removes water from the thin-layered liquid sourdough during contact. On the basis of various combinations of time and temperature and on the extent of the Maillard reaction, different type III sourdoughs are obtained, which show different degrees of caramelization or toasting and, as a consequence different flavouring activities. Since many volatile compounds (especially acetic acid) are missing due to the evaporation, even if to a different extent depending on the drying technique, pasteurization, cooling or salting can be applied to obtain a stabilized liquid or pasty sourdough (12, 48). With the exception of cooling, all the other stabilization systems lead to microbiota inactivation and stop gas and/or acid production (48), giving a sourdough which can

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be classified as type III. Especially liquid sourdough represents an advantage for industrial applications because it can be pumped and easily dosed, while showing constant quality (12). Generally, the use of a stabilized sourdough is quite simple. It can be stored at room temperature for a long time (30–60 days) and directly added to the final dough at a proportion of 5–10%. Because of the low cell density of lactic acid bacteria and yeasts, baker’s yeast is generally added to leaven the dough for bread production. Examples of applications of type III sourdough in the modern bakery industry are the use of a stabilized liquid rye sourdough for rye bread production; dextran-containing sourdough (produced by the use of selected L. mesenteroides strains) stabilized by cooling, pasteurization, or drying for the production of Panettone, rye bread and toast bread; pasteurized San Francisco sourdough in liquid form for the production of San Francisco bread (12). The selection of microbial strains represents the current challenge for sourdough bread-making industries and relies on several metabolic traits which are related to the most important flavour compounds. Overall, strains isolated from raw materials or sourdoughs are in vitro and in situ selected and evaluated mainly based on the kinetics of acidification, proteolytic activity and nitrogen metabolism and synthesis of aromatic compounds. Recently, fermentation processes based on selected lactic acid bacteria and liquid sourdough have been evaluated with the aim of improving the biosynthesis of specific chemical compounds (49).

4.8

Use of Pure Cultures of Sourdough Starters Versus Stabilized Sourdoughs

As for other fermented food biotechnologies (dairy, oenology, meat) the interest in using a commercial starter culture is rapidly increasing for sourdough breadmaking. In this context, some researches have been devoted to the use of freeze-dried or frozen preparations of lactic acid bacteria (4, 20). Nevertheless, only a few applications of selected lactic acid bacteria starters are currently documented both at artisan and industrial levels. Difficulties are mainly related to the possibility of obtaining highly concentrated lactic acid bacteria preparations as well as to the performance of the selected lactic acid bacteria in situ. From an applicative point of view starter cultures should be pre-cultivated in a mix of flour and water to obtain metabolically active strains before use as an inoculum. Nevertheless, it has been found that many strains, which were in vitro selected for interesting traits, show some difficulties in dominating the sourdough ecosystem and overcoming the endogenous lactic acid bacteria throughout refreshments. Recently, Siragusa et al. (24) demonstrated that, after ten consecutive refreshments, only three out of nine L. sanfranciscensis strains, which were in vitro selected for the optimal rate of acidification, proteolytic activity and release of flavour compounds, were able to persist during sourdough fermentation due to the dominance of autochthonous L. plantarum strains. Thus, the capability to adapt to the sourdough ecosystem seems to be an essential trait for selecting strains in order to obtain a sourdough with a constant microbial composition and

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performance. Currently, this seems the main limitation in the use of pure cultures as sourdough starters and the most innovative applications mainly deal with the use of stabilized sourdoughs.

References 1. Corsetti A, Farris GA, Gobbetti M (2010) Uso del lievito naturale. In: Gobbetti M, Corsetti A (eds) Biotecnologia dei prodotti lievitati da forno. Casa Editrice Ambrosiana, Milano, cap. 9 2. Onno B, Roussel P (1994) Technologie et microbiologie de la panification au levain. In: De Roissart H, Luquet FM (eds) Bactéries lactiques, vol 11. Lorica, Uriage, France, cap. V-5 3. Corsetti A, Settanni L (2007) Lactobacilli in sourdough fermentation: a review. Food Res Int 40:539–558 4. Hammes WP, Gänzle MG (1998) Sourdough breads and related products. In: Woods BJB (ed) Microbiology of fermented foods, vol 1. Blackie Academic and Professional, London, cap. 8 5. Wood BJB (2000) Sourdough bread. In: Robinson RK, Batt CA, Patel PD (eds) Encyclopedia of food microbiology, vol 1. Academic, London 6. De Vuyst L, Vancanneyt M (2007) Biodiversity and identification of sourdough lactic acid bacteria. Food Microbiol 24:120–127 7. Gobbetti M (1998) The sourdough microflora, interactions of lactic acid bacteria and yeasts. Trends Food Sci Tech 9:267–274 8. Kulp K (2003) Baker’s yeast and sourdough technologies in the production of U.S. bread products. In: Kulp K, Klaus L (eds) Handbook of dough fermentations. Marcel Dekker, New York, cap. 5 9. Ottogalli G, Galli A, Foschino R (1996) Italian bakery products obtained with sour dough: characterization of the typical microflora. Adv Food Sci 18:131–144 10. Böcker G, Stolz P, Hammes WP (1995) Neue Erkenntnisse zum Ökosysyem Sauerteig und zur Physiologie des sauerteig-typischen Stämme Lactobacillus sanfrancisco und Lactobacillus pontis. Getreide Mehl und Brot 49:370–374 11. Hammes WP (1991) Fermentation of non-dairy food. Food Biotech 5:293–303 12. Decock P, Cappelle S (2005) Bread technology and sourdough technology. Trends Food Sci Tech 16:113–120 13. Wiese BG, Strohmar W, Rainey FA, Diekmann H (1996) Lactobacillus panis sp. Nov., from sourdough with a long fermentation period. Int J Syst Bacteriol 46:449–453 14. Meroth CB, Walter J, Hertel C, Brandt MJ, Hammes WP (2003) Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:475–482 15. Meroth CB, Hammes WP, Hertel C (2003) Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient Gel electrophoresis. Appl Environ Microbiol 69:7453–7461 16. Pagani MA, Lucisano M, Mariotti M (2007) Traditional Italian products from wheat and other starchy flours. In: Hui YH (ed) Handbook of food products manufacturing. Wiley Interscience New York, USA, pp 327–388 17. Spicher G, Pomeranz Y (1985) Bread and other baked products. In: Ullmann’s Encyclopedia of industrial chemistry, vol A4, 5th edn. Verlag VCH, Weinheim 18. Vandamme EJ, Renard CEFG, Arnaut FRJ, Vekemans NMF, Tossut PPA (1997) Process for obtaining improved structure build-up of baked products. EP 0 790 003 B1 19. Corsetti A, Settanni L, Valmorri S, Mastrangelo M, Suzzi G (2007) Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations. Food Microbiol 24:592–600 20. De Vuyst L, Neysens P (2005) The sourdough microflora: biodiversity and metabolic interactions. Trends Food Sci Tech 16:43–56

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21. Vrancken G, Rimaux T, Weckx S, Leroy F, De Vuyst L (2011) Influence of temperature and backslopping time on the microbiota of a type I propagated laboratory wheat sourdough fermentation. Appl Environ Microbiol 77:2716–2726 22. Minervini F, Lattanzi A, De Angelis M, Di Cagno R, Gobbetti M (2012) Influence of artisan bakery- or laboratory-propagated sourdoughs on the diversity of lactic Acid bacterium and yeast microbiotas. Appl Environ Microbiol 78:5328–5340 23. Minervini F, De Angelis M, Di Cagno R, Pinto D, Siragusa S, Rizzello CG, Gobbetti M (2010) Robustness of Lactobacillus plantarum starters during daily propagation of wheat flour sourdough type I. Food Microbiol 27:897–908 24. Siragusa S, Di Cagno R, Ercolini D, Minervini F, Gobbetti M, De Angelis M (2009) Taxonomic structure and monitoring of the dominant population of lactic acid bacteria during wheat flour sourdough type I propagation using Lactobacillus sanfranciscensis starters. Appl Environ Microbiol 75:1099–1109 25. Kline L, Sugihara TF (1971) Microorganisms of the San Francisco sour dough bread process. II. Isolation and characterization of undescribed bacterial species responsible for the souring activity. Appl Microbiol 21:459–465 26. Corsetti A, Lavermicocca P, Morea M, Baruzzi F, Tosti N, Gobbetti M (2001) Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy. Int J Food Microbiol 64:95–104 27. Corsetti A, Settanni L, Van Sinderen D, Felis GE, Dellaglio F, Gobbetti M (2005) Lactobacillus rossii sp. nov. isolated from wheat sourdough. Int J Syst Evol Microbiol 55:35–40 28. Meroth CB, Hammes WP, Hertel C (2004) Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp. nov. Syst Appl Microbiol 27:151–159 29. Kleynmans U, Heinzl H, Hammes WP (1989) Lactobacillus suebicus sp. nov., an obligately heterofermentative Lactobacillus species isolated from fruit mashes. Syst Appl Microbiol 11:267–271 30. Vogel RF, Böcker G, Stolz P, Ehrmann M, Fanta D, Ludwig W, Pot B, Kersters K, Schleifer KH, Hammes WP (1994) Identification of lactobacilli from sourdough and description of Lactobacillus pontis sp. nov. Int J Syst Bacteriol 44:223–229 31. Rossi J (1996) The yeasts in sourdough. Adv Food Sci 18:201–211 32. Garofalo C, Silvestri G, Aquilanti L, Clementi F (2008) PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone. J Appl Microbiol 105:243–254 33. Gatto V, Torriani S (2004) Microbial population changes during sourdough fermentation monitored by DGGE analysis of 16 S and 26 S rRNA gene fragments. Ann Microbiol 54:31–42 34. Pulvirenti A, Solieri L, Gullo M, De Vero L, Giudici P (2004) Occurence and dominance of yeast species in sourdough. Lett Appl Microbiol 38:113–117 35. Vernocchi P, Valmorri S, Gatto V, Torriani S, Gianotti A, Suzzi G, Guerzoni ME, Gardini F (2004) A survey on yeast microbiota associated with an Italian traditional sweet-leavened baked good fermentation. Food Res Int 37:469–476 36. Iacumin L, Cecchini F, Manzano M, Osualdini M, Boscolo D, Orlic S, Comi G (2009) Description of the microflora of sourdoughs by culture-dependent and culture-independent methods. Food Microbiol 26:128–135 37. Hammes WP, Vogel RF (1995) The genus Lactobacillus. In: Woods BJB, Holzapfel WH (eds) The genera of lactic acid bacteria. Blackie Academic and Professional, London, pp 19–54 38. Corsetti A, De Angelis M, Dellaglio F, Paparella A, Fox PF, Settanni L, Gobbetti M (2003) Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses. J Appl Microbiol 94:641–654 39. Ricciardi A, Parente E, Piratino P, Paraggio M, Romano P (2005) Phenotypic characterization of lactic acid bacteria from sourdoughs for Altamura bread produced in Apulia (Southern Italy). Int J Food Microbiol 98:63–72 40. De Angelis M, Di Cagno R, Gallo G, Curci M, Siragusa S, Crecchio C, Parente E, Gobbetti M (2007) Molecular and functional characterization of Lactobacillus sanfranciscensis strains isolated from sourdoughs. Int J Food Microbiol 114:69–82

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41. Torriani S, Felis GE, Dellaglio F (2001) Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol 67:3450–3454 42. Gänzle MG, Vogel RF (2003) Contribution of reutericyclin to the stable persistence of Lactobacillus reuteri in an industrial sourdough fermentation. Int J Food Microbiol 80:31–45 43. Zapparoli G, Torriani S, Dellaglio F (1998) Differentiation of Lactobacillus sanfranciscensis strains by randomly amplified polymorphic DNA and pulsed-field gel electrophoresis. FEMS Microbiol Lett 166:324–332 44. Barnett JA, Payne RW, Yarrow D (2000) Yeast: characteristics and identification, 3rd edn. Cambridge University Press, Cambrige 45. Fernandez-Espinar MT, Martorell P, de Llanos R, Querol A (2006) Molecular methods to identify and characterize yeasts in foods and beverages. In: Yeasts in food and beverages. Springer, Berlino, pp 55–82 46. Onno B (1994) Les levains. In: Guinet R, Godin B (eds) La panification française. Lavoisier, Paris, cap. 8 47. Gobbetti M, De Angelis M, Corsetti A, Di Cagno R (2005) Biochemistry and physiology of sourdough lactic acid bacteria. Trends Food Sci Tech 16:57–69 48. Brandt MJ (2007) Sourdough products for convenient use in baking. Food Microbiol 24:161–164 49. Carnevali P, Ciati R, Leporati A, Paese M (2007) Liquid sourdough fermentation: industrial application perspectives. Food Microbiol 24:150–154 50. Collar C, Benedito de Barber C, Martínez-Anaya MA (1994) Microbial sourdoughs influence acidification properties and bread-making potential of wheat dough. J Food Sci 59:629–633 51. De Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135

Chapter 5

Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria Geert Huys, Heide-Marie Daniel, and Luc De Vuyst

5.1 Taxonomy of Sourdough Yeasts and Lactic Acid Bacteria 5.1.1

Taxonomy of Sourdough Yeasts

Yeasts are microscopic fungi that undergo typical vegetative growth by budding or fission resulting in an unicellular appearance and a sexual reproduction without a within or upon which the resulting spores are formed [1]. From the agro-alimentary and scientific point of view, yeasts are among the most important eukaryotes. Yeast species found in sourdough microbial communities share an adaptation to the specific and stressful environment created mainly by a low pH, high carbohydrate concentrations and high cell densities of lactic acid bacteria (LAB). Such adaptations can be found in species located mostly on one branch of the evolutionary tree

G. Huys BCCM/LMG Bacteria Collection and Laboratory for Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium H.-M. Daniel Mycothèque de l’Université catholique de Louvain (MUCL), Earth and Life Institute, Applied Microbiology, Mycology, Université catholique de Louvain, Croix du Sud 3 bte 6, 1348 Louvain-la-Neuve, Belgium L. De Vuyst (*) Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium e-mail: [email protected]

M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, DOI 10.1007/978-1-4614-5425-0_5, © Springer Science+Business Media New York 2013

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of fungi, which accommodates the ascomyceteous yeasts. Within this branch, recognized by the current classification in the phylum Ascomycota, as the subphylum Saccharomycotina, class Saccharomycetes, order Saccharomycetales [2], sourdough yeasts belong to different genera. The major sourdough yeasts belong to genera that are currently placed in the family Saccharomycetaceae, although family assignment of yeast genera is still difficult because of a lack of informative data. Basidiomycetous yeasts and dimorphic ascomycetes, also adapted to growth in liquid environments by unicellular growth forms, lack the fermentative abilities that are common to the Saccharomycetales and that are important for growth under oxygen limitations. The taxonomy of the Saccharomycetales, classically based on morphology and physiology, is in the process of being adapted to the increasing knowledge of evolutionary relationships reconstructed from gene sequences, in other words, a phylogenetic system of classification is being developed [2]. This implies a number of name changes. The new genus names have the advantage of reflecting the common genetic background of related yeast species, hereby providing an informative classification in contrast to the former largely artificial classification. An overview of recent name changes restricted to species that have been obtained from sourdough is given here. The name changes most relevant to the yeasts found in sourdough concern the genera Saccharomyces Meyen ex Reess and Pichia E.C. Hansen emend. Kurtzman. The genus Saccharomyces has been limited to the group of species known as Saccharomyces sensu stricto, including the type species of the genus, Saccharomyces cerevisiae, on the basis of multiple gene sequences [3]. The group of species formerly often addressed as Saccharomyces sensu lato has been divided into several genera. The new genus Kazachstania is accommodating the former Saccharomyces exiguus, Saccharomyces unisporus, and Saccharomyces barnettii as Kazachstania exigua, Kazachstania unispora, and Kazachstania barnettii, respectively. Saccharomyces kluyveri has been assigned to the new genus Lachancea as Lachancea kluyveri. The genus Pichia has been restricted to species closely related to the generic type species Pichia membranifaciens, including Pichia fermentans [4]. The former genus Issatchenkia has been integrated into the newly defined genus Pichia as its species are located on the same branch as the type species P. membranifaciens on the phylogenetic tree based on multiple gene sequences used for the redefinition of genera. While the species epithet of Issatchenkia occidentalis has been preserved in its new name Pichia occidentalis, a complete name change of Issatchenkia orientalis to Pichia kudriavzevii was necessary as the combination Pichia orientalis had been used for a different species before. The former species Pichia anomala and Pichia subpelliculosa were found to be only distantly related to the generic type species P. membranifaciens and have therefore been assigned to the newly created genus Wickerhamomyces as Wickerhamomyces anomalus and Wickerhamomyces subpelliculosus. A review of the taxonomic considerations including the earlier genus name Hansenula has been given by Kurtzman [5, 6]. Other, only occasionally from sourdough isolated species from the former genus Pichia have been reassigned to new genera, while preserving their species epithet and include Kodamaea ohmeri, Meyerozyma guilliermondii, Millerozyma farinosa, Ogataea polymorpha, Saturnispora saitoi, and Scheffersomyces stipitis.

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To determine the entities, or taxa,1 that deserve attention in the sourdough context, about 40 original publications were reviewed and the repeatedly reported species are listed in Table 5.1. These publications span the time from the early 1970s until present and it is obvious that not all of them are based on identification techniques that are currently considered as the most accurate. However, most of the six regularly (seven or more reports) encountered species S. cerevisiae (syn.2 S. fructuum); Candida humilis (syn. Candida milleri); P. kudriavzevii (syn. I. orientalis, anamorph Candida krusei); K. exigua (syn. S. exiguus, anamorph Candida [Torulopsis] holmii); Torulaspora delbrueckii, anamorph Candida colliculosa; and W. anomalus (syn. P. anomala, Hansenula anomala, anamorph Candida pelliculosa) can be distinguished from each other reasonably well by classical methods based on morphology and physiology as used in the reviewed literature up to the late 1990s. However, comparing studies using phenotypic identification techniques (n = 19) with those using DNA-based techniques (n = 23), the incidence of C. humilis has increased markedly in the DNA-based studies, probably at the cost of a decreased frequency of detecting K. exigua. These two phylogenetically closely related species may be mistaken for each other if using phenotypical identification methods. For example, originally S. exiguus was reported from San Francisco sourdough [7], while some strains from this study that were preserved in culture collections, later served for the description of C. milleri, currently a synonym of C. humilis. The sourdough isolate M14 reported as S. exiguus and deposited also as CBS 7901 was suggested to belong to C. humilis after DNA-based analyses [29, 50]. The regularity with which S. cerevisiae, C. humilis, P. kudriavzevii, K. exigua, T. delbrueckii, and W. anomalus are encountered in sourdough is an indication of their common association with this substrate. This is not necessarily an exclusive association with sourdoughs and some of the species have to be considered as generalists, able to thrive in a wide range of environmental conditions, as for example W. anomalus [51], while a specific ecological niche of the most frequently encountered species S. cerevisiae has been elucidated [52]. Other species are less frequently detected in sourdough, namely Candida glabrata; P. membranifaciens (anamorph Candida valida); Candida parapsilosis; Candida tropicalis; Candida stellata (syn. Torulopsis stellata); K. unispora (syn. S. unisporus, Torulopsis unisporus); Kluyveromyces marxianus (anamorph Candida kefyr); M. guilliermondii (syn. Pichia guilliermondii, anamorph Candida guilliermondii); and Saccharomyces pastorianus. Finally, 14 species have been mentioned only in single reports and may be considered as rather transient or present fortuitously in the sourdough ecosystem (Table 5.2).

1 A taxon (plural taxa) refers to a group of individuals that are judged to form a single unit. A taxon may or may not be given name or rank (species, genus, family, etc.). Primarily, the term serves communication about taxonomic units without the necessity or the possibility to be more specific about them. 2 Synonymous names and those of asporogenic forms (asexual or anamorphic forms, not producing ascospores) are only selectively mentioned if used in the sourdough literature. It is preferable to use the name of the sporogenous form (sexual or teleomorphic form) if it exists and if no strong reasons require the explicit referral to the asporogenous form.

Wheat (W), rye (R), sorghum (S), barley (B), maize (M), spelt (Sp), buckwheat (Bw), teff (T)

W W R W R W W, R S

Reference

[7] [8] [9] [10] [11] [12] [13] [14]

Number of analyzed sourdoughs

5 3 2 3 20 2 10 2

Phenotypic (P) or molecular (M) identification

P P P P P P P P

USA Italy Germany Spain Finland Spain Italy Sudan

Country

Saccharomyces cerevisiae syn. S. fructuum

d

11 X 11 29 X

Candida milleri syn. of C. humilis x

148a

Pichia kudriavzevii syn. Issatchenkia orientalis Anamorph C. krusei x

27

Kazachstania exigua syn. S. exiguus Anamorph C. (Torulopsis) holmii x

6

x 4

Wickerhamomyces anomalus syn. P. anomala, Hansenula anomala Anamorph C. pelliculosa 2

Candida stellata syn. T. stellata x

1

Kazachstania unispora syn. S. unisporus, T. unisporus Kluyveromyces marxianus Anamorph C. kefyr 1

Meyerozyma guilliermondii syn. P. guilliermondii Anamorph C. guilliermondii 6

Saccharomyces pastorianus Candida tropicalis

Candida parapsilosis

Pichia membranifaciens Anamorph C. valida Candida glabrata

Torulaspora delbrueckii Anamorph C. colliculosa

Candida humilis

Table 5.1 Overview of original studies that analyzed the yeast diversity of sourdoughs. Only species repeatedly obtained from sourdough are listed. Numbers in species columns indicate the number of obtained isolates. These numbers depend on the isolation method (i.e., one or multiple representatives of the same colony morphotype) and are mostly of value to judge species abundance within a given study. An “x” was used if the isolate number was not specified. Multiple x’s indicate the relative abundance of a detected species if mentioned. Studies that report on more than one sourdough may include sourdoughs of differing yeast species composition. However, a more detailed assignment of yeast species to single sourdoughs was not possible as not all studies reported the necessary data. Synonyms or asporogenic forms (anamorphs) are listed only if used in the cited references or if resulting from recent taxonomic changes

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[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41]

W B, M W M W R, M, W W R R, M W R W n.s. W R n.s. W W W n.s. W W n.s. W W W, R, Sp W

12 10 24 10 7 33 10 5 n.s. 5 2 25 14 1 3 35 13 8 1 35 1 1 7 10 3 33 4

P P P P P P P P, M P P, M P P P, M M P, M P, M P, M P, M M P, M P, M P, M M P, M M P, M M

France Morocco Italy Ghana Italy Portugal Italy Finland Portugal Greece Denmark Italy Italy Italy Germany Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Belgium Italy

48 118 58d 6d 6d 102 45 13 23 37 x 102d 60d

d

220 21d 51 xd 25 27 xd 3 7 14 xd 17 33d

18

27 36

2

15 xx

48 36

6

2 7 403 36 42 7

10c

23b

8

50 8 3

1 8

11

13 x

3

3

1

xx

12

25

2

5

1

13

25

177

2

5

1

27

62

2

19 3

25

12

x

1

1

x

(continued)

2

1

5 Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria 109

W W W W W

Reference

[42] [43] [44] [45] [46] 4 9 15 10 20

Table 5.1 (continued)

Wheat (W), rye (R), sorghum (S), barley (B), maize (M), spelt (Sp), buckwheat (Bw), teff (T) Number of analyzed sourdoughs

P, M P, M P P, M P, M

Phenotypic (P) or molecular (M) identification

Thailand Italy Pakistan Greece Italy

Country

4d 33 x 4d 49d

Saccharomyces cerevisiae syn. S. fructuum

3

Candida humilis

10

Candida milleri syn. of C. humilis

1

1

Pichia kudriavzevii syn. Issatchenkia orientalis Anamorph C. krusei

2

Kazachstania exigua syn. S. exiguus Anamorph C. (Torulopsis) holmii

163 5

Torulaspora delbrueckii Anamorph C. colliculosa Wickerhamomyces anomalus syn. P. anomala, Hansenula anomala Anamorph C. pelliculosa Candida glabrata Pichia membranifaciens Anamorph C. valida 1

Candida parapsilosis Candida stellata syn. T. stellata

1

Candida tropicalis Kazachstania unispora syn. S. unisporus, T. unisporus Kluyveromyces marxianus Anamorph C. kefyr Meyerozyma guilliermondii syn. P. guilliermondii Anamorph C. guilliermondii Saccharomyces pastorianus

G. Huys et al.

110

W

[48]

7

2

M

M

China

Ireland

43

xd 22

2

2

6

3

x 2

1

Total occurrences 38 19 14 12 8 7 4 3 2 2 2 2 2 2 2 a Originally designated as S. exiguus, but in culture collections deposited representative strains were reassigned to C. humilis. One of them served as the type strain for the description of the currently not taxonomically recognized population C. milleri (NRRL Y-7244, NRRL Y-7245, NRRL Y-7246, NRRL Y-7248; [49]). C. humilis and C. milleri isolates are listed here in partially separated columns as some studies distinguished them despite their taxonomic synonymy b Labeled as C. milleri by the authors, ITS sequence of one strain deposited as CBS 7541 shows intermediate similarities to C. humilis and C. milleri, while its ITS RFLP HaeIII type is similar to C. humilis c Retrospective identification based on the presence of type and reference strains [22] d No baker’s yeast added as explicitly mentioned by the authors n.s. Not specified by the original authors

Bw, T

[47]

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Table 5.2 Yeast species isolated from sourdough mentioned by single reports. None of these reports refer to these species as the sole or dominating yeast found in sourdough. Species considered as rare contaminants (e.g., Schizosaccharomyces pombe, Rhodotorula glutinis, R. mucilaginosa, Endomycopsis fibulinger) or reported as unidentified were not included Species Synonymsa Reference [12] Candida boidinii Candida parapsilosis [42] Hanseniaspora uvarum [38] Kodamaea ohmeri Pichia ohmeri [23] Lachancea kluyveri Saccharomyces kluyveri [20] Meyerozyma guilliermondii P. guilliermondii, anamorph C. guilliermondii [12] Ogataea polymorpha P. polymorpha [10] Pichia fermentans Anamorph C. lambica [31] Pichia occidentalis Issatchenkia occidentalis [20] Saccharomyces bayanus Saccharomyces inusitatus [7] Saturnispora saitoi P saitoi [9] Scheffersomyces stipitis P. stipitis [42] Wickerhamomyces P. subpelliculosa, Hansenula subpelliculosa [10] subpelliculosus Yarrowia lipolytica [24] a Synonyms or asporogenic forms (anamorphs) were listed only if used in the cited references or if resulting from recent taxonomic changes

The species complex C. humilis/C. milleri frequently detected in sourdough deserves a detailed elucidation. In terms of classification it was placed in the artificial genus Candida, because no sexual reproduction could be observed. Phylogenetically, C. humilis/C. milleri belongs to the same group as the genus Kazachstania [3]. It has, however, not yet been taxonomically placed in this genus, as the phylogenetic reclassification is treating sexually reproducing taxa with priority. The species C. humilis has been described based on a yeast strain associated with South African bantu beer, made from kaffir corn (Sorghum caffrorum) or finger millet (Eleusine coracana) [53]. The species C. milleri has been described to accommodate yeast strains isolated from San Francisco sourdough fermentations and initially assigned to S. exiguus [7, 49]. The basis for this reassignment were significantly higher guanine-plus-cytosine contents in selected San Franscisco sourdough strains compared to the type and other reference strains of S. exiguus and growth stimulation of C. milleri by calcium pantothenate. C. humilis and C. milleri were indicated to be conspecific based on their identical D1/D2 region large subunit (LSU) ribosomal DNA (rDNA) sequences, a locus that in rare cases may not suffice to resolve closely related species [54, 55]. DNA-DNA reassociation of at least 90% between the C. milleri type strain CBS 6897 and strain CBS 2664, the type strain of T. holmii var. acidilactici, a synonym of C. milleri, are of interest in this situation and confirms the conspecificity of these strains [54, 56]. Strain CBS 2664 shows ten substitutions in the internal transcribed spacer (ITS) regions of the rDNA, if compared to the type strain of C. milleri, indicating the degree of ITS divergence within this species.

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In the sourdough context, C. humilis is often distinguished from its synonym C. milleri by targeting the ITS rDNA region [27]. This is done by restriction fragment length polymorphisms (RFLPs) of the ITS generated by the restriction enzyme HaeIII that is recognizing and cutting the nucleotide sequence GGCC. This site is made unrecognizable by a single nucleotide change from C to T (resulting in GGCT) in a population represented by the C. milleri type strain. As a result of this single nucleotide substitution the RFLP analysis shows two fragments, in contrast to three fragments for strains with the intact recognition site. A comparison of relevant publicly available ITS sequences (CBS 5658: AY046174, CBS 6897: AY188851, SY13: DQ104399; CBS 2664 and CBS 7541: yeast database at www.cbs.knaw.nl/) shows the transitional position of some strains between the type strain sequences of C. humilis and C. milleri, especially CBS 7541, indicative of a continuum of ITS sequence variants between both type strains. Therefore, these two taxa might be best considered as populations of one species. Eventual heterogeneity of the species C. humilis, especially of applied value, should be documented and accompanied by the deposition of the isolates in public culture collections for further study.

5.1.2

Taxonomy of Sourdough Lactic Acid Bacteria

LAB comprise a heterogeneous group of Gram-positive, nonsporulating, strictly fermentative lactic acid-producing bacteria that play an important role in the organoleptic, health-promoting, technological, and safety aspects of various fermented food products. As a result of natural contamination through the flour or the environment or by deliberate introduction via dough ingredients, a wide taxonomic range of LAB has also been found in sourdoughs. In sourdough environments, LAB live in association with yeasts and are generally considered to contribute most to the process of dough acidification, while yeasts are primarily responsible for the leavening. Although also obligately homofermentative LAB have been isolated from sourdoughs, obligately or facultatively heterofermentative LAB species have the best potential and competitiveness to survive and grow in this particular food environment [57, 58]. Initially, classification of LAB was based on morphology, ecology, and physiological characteristics [59]. At a later stage, also chemotaxonomical properties such as cellular fatty acid and cell wall composition were included. In LAB as well as in many other bacterial groups, phenotypic characters are often limited in their taxonomic usefulness for discrimination of closely related species and suffer from poor interlaboratory exchangeability. As a result, differentiation of LAB solely based on phenotypic traits is generally only considered reliable at the genus level. The introduction of DNA-based techniques such as genomic mol % GC, DNA-DNA hybridization, and sequencing of ribosomal RNA (rRNA) genes has brought significant changes to LAB taxonomy [60–62]. Especially the use of rRNA gene sequences as evolutionary chronometers has allowed the elucidation of phylogenetic relationships between LAB species. As a result, comparison of 16S rRNA gene sequences

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with sequences in public online databases has become a standard approach for identification of unknown LAB isolates. However, the low evolutionary rate of ribosomal genes may compromise differentiation between LAB species exhibiting identical or nearly identical 16S rRNA gene sequences [63–66]. Alternatively, the use of multiple housekeeping genes encoding essential cellular functions has been proposed for sequence-based identification of LAB. For instance, classification of Lactobacillus species based on sequence analysis of the housekeeping genes pheS and rpoA proved to be highly congruent with 16S rRNA gene phylogeny [67]. The LAB species diversity associated with sourdoughs has been reviewed by several authors in recent years [57, 58, 68–70]. On the basis of these reviews, an updated overview of the LAB species most commonly found in fermented sourdough is compiled in Table 5.3. As is the case for many food ecosystems, this overview again highlights that lactobacilli are by far the most frequently recovered LAB species from sourdough ecosystems. The taxonomy of the genus Lactobacillus is extremely complex; according to the April 2011 update, at least 171 species names have so far been proposed in this genus (www.bacterio.cict.fr/l/lactobacillus.html). However, as several of the proposed species names have meanwhile been synonymized, the actual number of phylogenetically unique species is lower. In sourdoughs, more than 55 Lactobacillus species have been identified, of which the large majority are obligately heterofermentative (Table 5.3). Given the taxonomic complexity of this genus, accurate identification of unknown Lactobacillus isolates requires specific expertise, for example the use of methods that offer sufficient taxonomic resolution and the correct interpretation of identification results by comparison with complete and up-to-date databases. In most of the older studies, however, identification of lactobacilli mainly or even exclusively relied on phenotypic approaches with limited taxonomic resolution at species level. Therefore, it is safe to assume that some of the Lactobacillus species previously reported in sourdough environments may have been incorrectly identified at the species or even at the genus level. A typical example is the taxonomic situation in the Lactobacillus plantarum group where discrimination between the ubiquitous sourdough bacterium Lb. plantarum and the phylogenetically highly related Lactobacillus paraplantarum and Lactobacillus pentosus may be problematic when identification methods with insufficient taxonomic resolution are used. In this regard, Torriani and colleagues [71] were among the first to suggest that sequences of housekeeping genes such as recA rather than 16S rRNA gene sequences are recommended to distinguish between members of this phylogenetically tight species group. Likewise, several sourdough isolates initially assigned to Lactobacillus alimentarius may in fact belong to the later described and closely related Lactobacillus paralimentarius due to phenotypic misidentification [72]. Also in this case, it has been shown that molecular fingerprint- or sequence-based methods are required to differentiate between both species [67, 73]. In Lactobacillus rossiae, a remarkable intraspecific heterogeneity leading to the identification of several subspecific clusters based on pheS gene sequencing may complicate unambiguous identification of Lb. rossiae strains [77]. Finally, nomenclatural issues may also be a cause for taxonomic confusion. Corrections of originally misspelled specific epithets, such as “Lactobacillus

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Table 5.3 LAB species generally associated with sourdough fermentation or found in fermented sourdoughsa Facultatively Obligately Obligately heterofermentative homofermentative heterofermentativeb Lb. acidifarinae Lb. brevis Lb. buchneri Lb. cellobiosus Lb. collinoides Lb. crustorum Lb. curvatus Lb. fermentum Lb. fructivorans

Lb. alimentarius Lb. casei/paracasei Lb. coleohominis Lb. kimchi Lb. paralimentarius Lb. pentosus Lb. perolens Lb. plantarum Lb. sakei

Lb. frumenti Lb. hammesii Lb. hilgardii Lb. homohiochii Lb. kefiri Lb. kunkeei Lb. lindneri Lb. mucosae Lb. namurensis Lb. nantensis Lb. nodensis Lb. oris Lb. panis Lb. parabuchneri Lb. pontis Lb. reuteri Lb. rossiae Lb. sanfranciscensis Lb. secaliphilus Lb. siliginis Lb. spicheri Lb. vaginalis Lb. zymae Le. citreum Le. gelidum Le. mesenteroides subsp. cremoris Le. mesenteroides subsp. dextranicum Le. mesenteroides subsp. mesenteroides W. cibaria W. confusa

P. acidilactici P. dextrinicus P. pentosaceus

E. casseliflavus E. durans E. faecalis E. faecium Lb. acidophilus Lb. amylolyticus Lb. amylovorus Lb. crispatus Lb. delbrueckii subsp. delbrueckii Lb. farciminis Lb. gallinarum Lb. gasseri Lb. helveticus Lb. johnsonii Lb. mindensis Lb. nagelii Lb. salivarius Lc. lactis subsp. lactis S. constellatus S. equinus S. suis

(continued)

116 Table 5.3 (continued) Obligately heterofermentativeb

G. Huys et al.

Facultatively heterofermentative

Obligately homofermentative

W. hellenica W . kandleri W. paramesenteroides W. viridescens E. Enterococcus, Lb. Lactobacillus, Lc. Lactococcus, Le. Leuconostoc, P. Pediococcus, S. Streptococcus, W. Weissella a Data compiled from [57, 58, 68, 70] b Classification in glucose fermentation types according to Felis and Dellaglio [59]

sanfrancisco” [75] (now Lactobacillus sanfranciscensis) and “Lactobacillus rossii” [76] (now Lb. rossiae), and the synonymization of species, such as the recognition of Lactobacillus suntoryeus as a synonym of Lactobacillus helveticus [77], can take a while to be introduced in subsequent taxonomic literature. Triggered by the introduction of molecular DNA-based taxonomic methods in sourdough microbiology and the growing number of 16S rRNA gene sequences in public databases, in recent years many new Lactobacillus species have been described which were first isolated from a sourdough environment. Since 2000, 13 new Lactobacillus species originally isolated from sourdoughs have been proposed, i.e., Lactobacillus frumenti [78], Lactobacillus mindensis [79], Lactobacillus spicheri [80], Lactobacillus acidifarinae [81], Lactobacillus zymae [81], Lactobacillus hammesii [82], Lb. rossiae [76], Lactobacillus siliginis [83], Lactobacillus nantensis [84], Lactobacillus secaliphilus [85], Lactobacillus crustorum [86], Lactobacillus namurensis [87], and Lactobacillus nodensis [88] (Table 5.3). However, many of these species have only been reported once or are rarely isolated from this type of fermented food, and are represented by only a few strains. Therefore, it is not clear which of these species are really typical for sourdough environments and if so, what their geographical distribution is. In fact, only a few Lactobacillus species such as the obligately heterofermentative Lb. sanfranciscensis and the facultatively heterofermentative Lb. paralimentarius seem to be optimally adapted to this specific environment and are rarely isolated from other sources. Other heterofermentative species such as Lactobacillus brevis and the facultatively heterofermentative Lb. plantarum are also frequently isolated from fermented sourdoughs, but have also been found in many other food and nonfood environments [69]. Although the LAB microbiota of sourdoughs is clearly dominated by lactobacilli, other less predominant or subdominant LAB species may also be found, including members of the genera Weissella, Pediococcus, Leuconostoc, Lactococcus, Enterococcus and Streptococcus (Table 5.3). Of these, specific species of Weissella, Pediococcus, and Leuconostoc are particularly well adapted to survive and grow in plant-derived materials [57, 89]. The taxonomy of the latter three genera is much less complex than in Lactobacillus, and their presence in sourdoughs is restricted to only a few species. Weissellas are obligately heterofermentative LAB of which a

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number of species produce dextran, the best-documented exopolysaccharide formed by heterofermentative LAB. In sourdoughs, the dextran-producing species Weissella cibaria and Weissella confusa are most frequently found. Both taxa are positioned together on one of the four phylogenetic branches in this genus based on 16S rRNA gene analyses [90], but can be differentiated using restriction analysis of the amplified 16S rDNA [91], randomly amplified polymorphic DNA-PCR (RAPDPCR) [92], and PCR targeting the ribosomal ITS [26]. Within the facultatively heterofermentative pediococci, the species Pediococcus acidilactici and Pediococcus pentosaceus are most commonly found in sourdoughs. Differentiation between these two biochemically and phylogenetically related species can be achieved by fingerprinting methods such as ribotyping [93, 94], restriction analysis of the amplified 16S rDNA (16S-ARDRA) [95], RAPD-PCR [96, 97], and by sequence analyses of the 16S rRNA gene, the ribosomal ITS regions and the heat-shock protein 60 gene [98]. In the obligately heterofermentative genus Leuconostoc, the majority of sourdough isolates so far identified belong to Leuconostoc mesenteroides and Leuconostoc citreum. In the former species, further taxonomic distinction is made at subspecies level between Le. mesenteroides subsp. mesenteroides, Le. mesenteroides subsp. dextranicum, and Le. mesenteroides subsp. cremoris (www.bacterio.cict.fr/l/leuconostoc.html). The three subspecies can be separated by RAPD-PCR fingerprinting [99].

5.2 5.2.1

Microbial Species Diversity of Sourdoughs Influence of Geography

5.2.1.1 The Origin of Sourdough Historically, sourdough production started as a conditio sine qua non to process cereals for the production of baked goods [100]. Indeed, thousands of years ago the first bread production must have been based on spontaneous wild lactic acid fermentation whether or not associated with yeasts and with little or no leavening. Leavening could not have been very pronounced because of the use of barley (Hordeum vulgare) and ancient grains [such as spelt (Triticum aestivum subsp. spelta), emmer (Triticum turgidum subsp. dicoccum), and kamut (T. turgidum subsp. turanicum)] in early times and no addition of yeast for leavening. This form of flat (sour) bread production is still daily practice in many countries of the world, in particular in African countries and the Middle East. Leavening must have been an accidental discovery when yeasts from the air or the flour were allowed to ferment the cereal dough mixture extensively. However, it was only from the late nineteenth century onwards that yeast starter cultures were introduced for bread production from wheat (T. aestivum and Triticum durum) flour, first by using brewing yeasts as remnants of beer production followed by intentionally produced commercial bakers’ yeast, S. cerevisiae. Consequently, sourdough bread must have been consumed

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for a long time. Also afterwards, bread production in countries relying on cereals other than wheat such as rye (Secale cereale) had still to be supported by lactic acid fermentation. Rye bread baking requires dough acidification to inhibit the abundant a-amylase in the rye flour and to make rye starch and pentosans more water-retaining to form a good dough texture since not enough gluten is present in rye. Hence, various (rye) breads from Germany, Central European countries, and Scandinavia are based on sourdough. In the USA, sourdough was the main base of bread supply in Northern California during the California Gold Rush, because of the easy way to store it and to keep it active for daily bread production. Today, San Francisco sourdough bread is commercially produced in the San Francisco area and it remains a part of the culture of the San Francisco bay area. In the early 1970s, the responsible sourdough bacterium was identified as Lb. sanfrancisco, now Lb. sanfranciscensis, named according to the area where it was discovered [75]. Notice that this LAB species is actually identical to Lb. brevis var. lindneri (now Lb. sanfranciscensis), which was found to be responsible for various sourdough breads produced in Europe [17]. Also, it was shown that these sourdough LAB species occur in a stable association with the yeasts C. humilis (syn. C. milleri) and K. exigua (syn. S. exiguus), respectively [7, 101]. Nowadays, sourdough is used for its technological (dough processing and bread texture, flavor, and shelf life) and nutritional effects [57, 58, 69, 102, 103]. Moreover, sourdough products are appreciated for their traditional value, gastronomic quality, and natural and healthy status [104].

5.2.1.2 The Origin of Sourdough Variation Thanks to the fact that craftsmanship has determined bakery practice for a long time, a huge variety of bakery products, in particular those based on sourdough, exists, which may differ considerably from region to region. Most of these products, including breads, cakes, snacks, and pizzas, originate from very old traditions. For instance, in Italy, numerous different types of sourdough breads exist, often called according to the name of the region, such as Altamura bread and Pugliese bread [101]. Also, seasonal varieties exist, which are traditionally produced on the occasion of religious festivities. For instance, Panettone cake in Milan and Pandoro in Verona are made for Christmas, while Colomba is a Milanese cake made for Easter. Alternatively, sourdough-based products such as crackers, French baguettes, and Italian ciabattas are much more common, although the original sourdough recipe has been replaced by the faster growing bakers’ yeast that is in only few cases allowed to ferment longer to enable contaminating LAB to develop for flavor formation (pre-doughs or type 0 sourdoughs). Fortunately, numerous bakery sourdoughs have been kept alive for tens of years through backslopping procedures, i.e., repeated cyclic re-inoculation of a new batch of flour and water from a previous one with a so-called “sour” during refreshment of the flour-water mixture by the baker, thereby assuring the quality of the baked goods produced thereof. It turned out that these traditional sourdoughs harbor a mixture of distinctive yeast and LAB strains, which may be held responsible for the typical organoleptic quality of the breads

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made thereof, as backslopping results in a prevalence of the best-adapted strains [57, 68, 69]. This diversity of natural sourdough starters likely accounts for the variety of artisan sourdoughs produced by bakeries, whether or not typical for a certain geographical region. Alternatively, sourdough starter cultures, comprised of one or more defined strains, are commercially available now. In addition to Lb. sanfranciscensis strains, commercially available strains of other LAB species include Lb. brevis, Lactobacillus delbrueckii, Lactobacillus fermentum, and Lb. plantarum, albeit that not all strains in use are competitive enough to dominate the sourdough fermentation processes that have to be started up [69]. These starter cultures are used for rapid acidification of the raw materials and flavor formation upon fermentation. Also, industrial manufacturers produce dried sourdough powders that are used as nonliving flavor ingredients in industrial bread production. 5.2.1.3

Region-Specific Sourdoughs and Their Associated Microbiota

Whereas it was initially thought that a relationship could be seen between the presence of certain LAB species and the geographical origin of a particular sourdough, it turned out through systematic and detailed taxonomic investigations that the species diversity of both LAB and yeasts of local sourdoughs has nothing to do with the geography of the sourdough production process (Tables 5.1 and 5.4; 57, 68, 69). For instance, the typical sourdough bacterium of San Francisco sourdough bread of the San Francisco bay area, Lb. sanfranciscensis, has been found in various wheat sourdoughs throughout Europe, and hence its (unique) presence should be ascribed to other factors, which are mainly based on the fermentation technology and practical conditions applied [121, 152–154]. In various countries, such as in Italy, several studies have been focused on region-specific sourdoughs (Table 5.4). However, no clear-cut relationship could be shown between for the region typical sourdoughs and their associated microbiota. In contrast, Italian sourdoughs harbor simple to very complex communities of LAB species depending on the final products examined, among which Lb. brevis, Lb. (par) alimentarius, Lb. plantarum, Lb. sanfranciscensis, Lb. fermentum, P. pentosaceus, and W. confusa are widespread. Similarly, Belgian bakery sourdoughs have been analyzed extensively and are characterized by LAB consortia of Lb. brevis, Lb. hammesii, Lb. nantensis, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. sanfranciscensis, and/or P. pentosaceus [105, 106, 155]. Sourdoughs with both stable large and stable restricted species diversities may occur [106]. Also, Lb. rossiae seems to have a wide distribution in sourdoughs, as has been shown through its isolation from sourdoughs in Central and Southern Italy, Belgium, and elsewhere [74, 105, 106, 156–158]. LAB species are responsible for the acidification of the dough and contribute to flavor formation. Besides LAB species, a large variety of yeast species are found in sourdough ecosystems (Tables 5.1 and 5.2). S. cerevisiae has been found in almost every sourdough study (38 out of 42 reviewed) regardless as to whether or not bakers’ yeast is added (14 studies mention S. cerevisiae, although they indicate that no bakers’ yeast was added). A single sourdough usually harbors only one or two yeast species at a given time, among which C. humilis (and K. exigua) and P. kudriavzevii occur most frequently [40]. Yeasts are responsible for the leavening of the dough and also contribute to flavor formation.

Country Belgium

Wheat (laboratory) Molecular Spelt (laboratory) Molecular Wheat (laboratory) Molecular + microarray Spelt (laboratory) Molecular + microarray Rye (laboratory) Molecular

Spelt Molecular

Rye Molecular Wheat-rye Molecular

Method of identification Wheat Molecular

[107] [107] [108] [108] [109]

Lb. brevis, Lb. paraplantarum, Lb. plantarum, Lb. rossiae Lb. curvatus, Lb. fermentum, Lb. plantarum, P. pentosaceus Lb. fermentum, Lb. plantarum, P. pentosaceus, W. confusa Lb. brevis, Lb. fermentum, Lb. plantarum, P. pentosaceus

[105, 106]

[105, 106]

[105, 106]

Reference(s) [86, 87, 105, 106]

E. mundtii, Lb. brevis, Lb. curvatus, Lb. fermentum, Lb. paracasei, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb. sanfranciscensis, P. acidilactici, P. pentosaceus, W. confusa Lb. brevis, Lb. curvatus, Lb. hammesii, Lb. nantensis, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb. sakei, Lb. sanfranciscensis, P. pentosaceus, W. cibaria Lb. fermentum, Lb. plantarum

Lb. acidifarinae, Lb. brevis, Lb. buchneri, Lb. crustorum, Lb. hammesii, Lb. helveticus, Lb. namurensis, Lb. nantensis, Lb. parabuchneri, Lb. paracasei, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb. sakei, Lb. sanfranciscensis, Lb. spicheri, Leuc. mesenteroides, P. pentosaceus, W. cibaria, W. confusa Lb. brevis, Lb. hammesii, Lb. nantensis, Lb. paralimentarius, Lb. plantarum

LAB species reported

Table 5.4 Nonexhaustive overview of the species diversity of LAB in sourdoughs made from various flours and of different geographical origins. The method of identification of the LAB species is indicated Cereal flour type

120 G. Huys et al.

Germany

France

Finland

Wheat Phenotypical

Wheat Molecular Wheat Molecular

Wheat Phenotypical Wheat Molecular

Rye Phenotypical Teff (enjera) Phenotypical Rye Phenotypical Wheat Phenotypical

Denmark

Ethiopia

Wheat Molecular

Method of identification

China

Country

Cereal flour type

Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii, Lb. fermentum, Lb. plantarums

Lb. acidophilus, Lb. brevis, Lb. casei, Lb. curvatus, Lb. delbrueckii subsp. delbrueckii, Lb. plantarum, Leuc. mesenteroides subsp. dextranicum, Leuc. mesenteroides subsp. mesenteroides, P. pentosaceus Lb. brevis, Lb. curvatus, Lb. plantarum, Lb. rhamnosus, Leuc. mesenteroides, P. pentosaceus Lb. frumenti, Lb. hammesii, Lb. nantensis, Lb. panis, Lb. paralimentarius, Lb. pontis, Lb. sanfranciscensis, Lb. spicheri, Leuc. mesenteroides subsp. mesenteroides Lb. frumenti, Lb. hammesii, Lb. nantensis, Lb.panis, Lb. paralimentarius, Lb. sanfranciscensis, Lb. spicheri, Leuc. mensenteroides subsp. mensenteroides E. hirae, Lb. brevis, Lb. curvatus, Lb. paracasei, Lb. paraplantarum, Lb. pentosus, Lb. plantarum, Lb. sakei, Lb. sanfranciscensis, Lc. lactis, Leuc. citreum, Leuc. mesenteroides, P. pentosaceus, W. cibaria, W. confusa

E. faecalis, Lb. brevis, Lb. fermentum, Lb. plantarum, Leuc. mesenteroides, P. cerevisiae Lb. acidophilus, Lb. casei, Lb. plantarum

Lb. brevis, Lb. crustorum, curvatus, Lb. fermentum, Lb. guizhouensis, Lb. helveticus, Lb. mindensis, Lb. paralimentarius, Lb. plantarum, Lb. rossiae, Lb. sanfranciscensis, Lb. zeae, Lc. lactis, Leuc. citreum, W. cibaria, W. confusa Lb. amylovorus, Lb. panis, Lb. reuteri

LAB species reported

[116]

[115]

[114]

[113]

[112]

[111]

[11]

[110]

[25]

[48]

(continued)

Reference(s) 5 Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria 121

Country

Rice Phenotypic + molecular Wheat Molecular Rye Molecular

Wheat (Panettone, wheat bread from Germany, Italy, Sweden, and Switzerland) Phenotypical Rye Molecular Rye (bran) – use of a starter Molecular

Rye – use of a starter Phenotypical

Method of identification Rye Phenotypical

Table 5.4 (continued) Cereal flour type

[80]

Lb. crispatus, Lb. fermentum, Lb. frumenti, Lb. panis, Lb. pontis (rye, type II) Lb. johnsonii, Lb. reuteri (rye bran, type II) Lb. paracasei, Lb. paralimentarius, Lb. spicheri

[121]

[121]

[29]

Lb. mindensis, Lb. sanfranciscensis (rye, type I)

E. faecium, Lb. casei, Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis, P. pentosaceus, W. confusa Lb. pontis, Lb. sanfranciscensis

[120]

[119]

[118]

Reference(s) [9, 117]

Lb. amylovorus, Lb. frumenti, Lb. pontis/panis, Lb. reuteri (type II)

Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. buchneri, Lb.casei, Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. plantarum, Lb. sanfranciscensis Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. buchneri, Lb. casei, Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. plantarum, Lb. sanfranciscensis, Pediococcus spp. Lb. brevis, Lb. casei, Lb. farciminis, Lb. hilgardii, Lb. homohiochii, Lb. plantarum (spontaneous) Lb. brevis, Lb. hilgardii, Lb. sanfranciscensis,W. viridescens (masa madre)

LAB species reported

122 G. Huys et al.

Italy

Greece

Country

Wheat (Southern Italy) Molecular

Amaranth (from India, Peru, Mexico, Germany) Molecular Cereals and pseudocereals Molecular Wheat Phenotypical + molecular Wheat (panettone) Phenotypical Wheat (panettone, brioche) Phenotypical Wheat (Umbria) Phenotypical Pizza from Naples Phenotypical Wheat (Molise, Umbria, Venise) Molecular Mother sponges (Lombardy) Molecular Wheat (Apulia) Molecular

Method of identification

Cereal flour type

[13]

[17] [124] [125, 126]

[21]

[26]

Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis, Leuc. mesenteroides, Pediococcus spp. Lb. farciminis, Lb. plantarum, Lb. sanfranciscensis Lb. plantarum, Lb. sakei, Leuc. gelidum, Leuc. mesenteroides Lb. sanfranciscensis

Lb. sanfranciscensis

Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. delbrueckii subsp. delbrueckii, Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis, Lc. lactis subsp. lactis, Leuc. citreum, W. confusa Lb. alimentarius, Lb. brevis, Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. planta rum, Lb. sanfranciscensis, W. confusa

Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria (continued)

[127]

[8]

[72]

Lb. brevis, Lb. plantarum

Lb. brevis, Lb. paralimentarius, Lb. sanfranciscensis, Lb. zymae, W. cibaria

[123]

Lb. fermentum, Lb. helveticus, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. spicheri

Reference(s) [122]

Lb. plantarum, Lb. sakei, P. pentosaceus

LAB species reported

5 123

Country

Wheat (Abruzzo) Molecular Wheat (Panettone from Central Italy) Molecular Wheat (Cornetto, Basilicata) Phenotypic + molecular Wheat – use of a starter Phenotypic + molecular

Method of identification Wheat (Molise) Molecular Wheat (Sicily) Molecular Wheat (Molise) Molecular Wheat (Altamura, Apulia) Phenotypical + molecular Wheat (Sardinia) Molecular Wheat (Abruzzo) Physiological + molecular

Cereal flour type

Table 5.4 (continued)

[129] [130]

[131] [133]

Lb. brevis, Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis Lb. brevis, Lb. casei, Lb. paracasei, Lb. plantarum, Leuc. mesenteroides

Lb. brevis, Lb. pentosus, Lb. plantarum, Lb. sanfranciscensis, W. confusa Lb. alimentarius, Lb. brevis, Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. frumenti, Lb. hilgardii, Lb. mindensis, Lb. panis, Lb. paralimentarius, Lb. paraplantarum, Lb. pentosus, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb. sanfranciscensis E. faecium, P. pentosaceus

[39]

[134]

[41]

Lb. brevis, Lb. farciminis, Lb. plantarum, Lb. sanfranciscensis, Leuc. mesenteroides Lb. curvatus, Lb. paraplantarum, Lb. pentosus, Lb. plantarum, Leuc. mensenteroides, W. cibaria Lb. brevis, Lb. paralimentarius, Lb. plantarum, Lb. sanfranciscensis, Lc. lactis, Leuc. durionis, Leuc. fructosus, P. pentosaceus, W. cibaria

[133]

[128]

Reference(s) [33]

Lb. arizonensis/plantarum, Lb. brevis, Lb. kimchii/paralimentarius, Lb. paraplantarum, Lb. sanfranciscensis Lb. casei, Lb. kimchii/alimentarius, Lb. plantarum, Lb. sanfranciscensis

LAB species reported

124 G. Huys et al.

Ireland

Iran

Country

Wheat – use of a starter Molecular Barley – use of a starter Molecular Emmer Molecular Spelt Molecular Wheat Molecular Wheat germ Molecular Sangak Phenotypical Oat Molecular Buckwheat Molecular Buckwheat – use of a starter Molecular Teff Molecular

Wheat (Marche) Phenotypic + molecular

Method of identification

Cereal flour type Reference(s)

[137] [138] [139]

Lb. brevis, Lb. curvatus, Lb. plantarum, Lb. sanfranciscensis, P. pentosaceus/ acidilactici, W. confusa Lb. plantarum, Lb. rossiae, P. pentosaceus Lb. plantarum, Lb. rossiae, P. pentosaceus, W. confusa

Lb. fermentum, Lb. gallinarum, Lb. pontis, Lb. vaginalis, Leuc. holzapfelii, P. pentosaceus

[47]

Lb. crispatus, Lb. fermentum, Lb. gallinarum, Lb. graminis, Lb. plantarum, Lb. sakei, Lb. vaginalis, Leuc. holzapfelii, P. pentosaceus, W. cibaria Lb. amylovorus, Lb. brevis, Lb. fermentum, Lb. frumenti, Lb. paralimentarius, Lb. plantarum, Leuc. argentinum, W. cibaria

[47] (continued)

[142]

[141]

Lb. coryniformis, Leuc. argentinum, P. pentosaceus, W. cibaria

[140]

[137]

Lb. plantarum, Lb. rossiae, W. confusa

Lb. brevis, Lb. plantarum, Leuc. mesenteroides, P. cerevisiae

[136]

[135]

[43]

Lb. alimentarius, Lb. brevis, Lb. paralimentarius, Lb. plantarum

Lb. brevis, Lb. casei, Lb. curvatus, Lb. fermentum, Lb. paracasei, Lb. paralimentarius, Lb. plantarum, Lb. rhamnosus, Lb. sakei, Lb. sanfranciscensis, Leuc. citreum, Leuc. pseudomesenteroides, W. cibaria, W. confusa Lb. paralimentarius, Lb. plantarum, Lb. rossiae, Lb. sanfranciscensis, Lc. lactis subsp. lactis, P. pentosaceus, W. cibaria, W. cibaria/confusa

LAB species reported

5 Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria 125

Sudan

Spain

Russia

Portugal

Nigeria

Morocco

Mexico

Country

Maize (broa, Northern Portugal) Phenotypical Rye Phenotypical Wheat Phenotypical Wheat Phenotypical Sorghum (kisra) Phenotypical Kisra Molecular

Method of identification Teff – use of a starter Molecular Maize (pozol) Molecular Sourdough ferments, traditional starter sponges Phenotypical Soft wheat flour Phenotypical Maize

Table 5.4 (continued) Cereal flour type

[14] [148]

E. faecalis, Lb. fermentum, Lb. helveticus, Lb. reuteri, Lb. vaginalis, Lc. lactis

[12, 147]

Lb. brevis, Lb. cellobiosus, Lb. plantarum, Leuc. mesenteroides Lb. amylovorus, Lb. fermentum, Lb. reuteri

[10]

[146]

[21]

Lb. brevis, Lb. plantarum

Lb. brevis, Lb. fermentum, Lb. plantarum

[144]

Lb. buchneri, Lb. casei, Lb.delbrueckii, Lb. plantarum, Lb. sanfranciscensis, Leuc. mesenteroides, P. pentosaceus Lb. brevis, Lb. casei, Lb. fermentum, Lb. plantarum, Leuc. mesenteroides, P. acidilactici E. casseiliflavus, E. durans, E. faecium, Lb. brevis, Lb. curvatus, Lc. lactis subsp. lactis, Leuconostoc spp., S. constellantus, S. equines

[145]

[16]

[143]

Reference(s) [142]

Lb. brevis, Lb. buchneri, Lb. casei, Lb. plantarum, Leuc. mesenteroides, Pediococcus spp.

Lb. amylovorus, Lb. brevis, Lb. fermentum, Lb. frumenti, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. reuteri, Lb. sanfranciscensis, P. acidilactici Lb. alimentarius, Lb. casei, Lb. delbrueckii, Lb. lactis, Lb. plantarum, S. suis

LAB species reported

126 G. Huys et al.

Rye-wheat Phenotypical Rye Phenotypical Wheat Phenotypic + molecular Wheat Phenotypic Wheat Phenotypic Wheat Molecular

Method of identification

[75] [121]

Lb. paralimentarius, Lb. plantarum, Lb. sanfranciscensis

[151]

[42]

[150]

[149]

Reference(s)

C. divergens, Lb. acetotolerans, Lb. amylophilus, Lb. brevis, Lb. plantarum, Lb. sakei, P. acidilactici, P. pentosaceus, T. halophilus Lb. sanfranciscensis

Lb. casei, Lb. plantarum

Lb. acidophilus, Lb. brevis, Lb. delbrueckii, Lb. farciminis, Lb. fermentum, Lb. plantarum, Lb. rhamnosus, Lb. sanfranciscensis, W. viridescens Lactobacillus sp., P. pentosaceus

LAB species reported

C. Carnobacterium, E. Enterococcus, Lb. Lactobacillus, Lc. Lactococcus, Le. Leuconostoc, P. Pediococcus, S. Streptococcus, W. Weissella

USA

Turkey

Thailand

Sweden

Country

Cereal flour type

5 Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria 127

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Single isolations of yeast and LAB species have caused former misinterpretations of their association with certain sourdough-producing regions, not only because of the random isolation itself but also regarding the single habitat (sourdough) explored. Thus, the association of, for instance, Lb. spicheri with German rice sourdoughs [80] might represent accidental discoveries [57, 68, 69]. Instead, the dedicated use of basic raw materials as well as the technological procedures applied rather determines the stability and persistence of the yeast and LAB communities involved in the sourdough fermentation process. Indeed, the presence of Lb. sanfranciscensis in wheat sourdoughs, which was for a long time the sole habitat wherein this LAB species could be found [now, it has been detected in rye sourdoughs (Table 5.4) and the insect gut as well [159]], can be ascribed to its selection by the type of technology applied, i.e., backslopping practices, temperature of incubation of the dough, pH of the dough, and/or microbial interactions [113, 121, 135, 160, 161]. However, the use of certain raw materials, encompassing cereal types and other ingredients such as adjunct carbohydrates, salt, yoghurt, herbs, etc., and operational practices, such as dough yield and refreshment times, may be linked to local traditions, may favor particular microorganisms as a result of trophic and metabolic relationships and interactions (both cooperation and antibiosis), and hence may associate specific LAB and/or yeast species with specific geographical regions. For instance, the use of rye may select for amylase-positive homofermentative Lactobacillus amylovorus, although higher temperatures cause a shift toward the predominance of heterofermentative lactobacilli [120, 162]. The dominance of mainly heterofermentative LAB species in traditional sourdoughs is caused by a highly adapted carbohydrate metabolism, a dedicated amino acid assimilation, and environmental stress responses [57, 58, 68, 163–166]. In particular, maltose, as the most abundant fermentable energy source in dough, is metabolized via the maltose phosphorylase pathway and the pentose phosphate shunt by strictly heterofermentative LAB species such as Lb. sanfranciscensis, Lactobacillus reuteri, and Lb. fermentum. This efficient maltose metabolism coupled to the use of external alternative electron acceptors such as fructose, together with specific pathways such as the arginine deiminase (ADI) pathway, and various environmental stress responses such as response to acidic conditions, increases its competitiveness in the harsh sourdough environment [167–172]. Moreover, maltose-positive LAB species such as Lb. sanfranciscensis often form a stable association with maltose-negative yeast species such as C. humilis, thereby preventing competition for the same carbohydrate sources [58, 164, 165]. Also, the production of specific inhibitory compounds, maintained through backslopping, such as the antibiotic reutericyclin produced by Lb. reuteri, may favor the dominance of this LAB species, as is the case for certain German type II sourdoughs [173].

5.2.2

Influence of Cereals and Other Raw Materials

Cereal flours are not sterile. Their microbiological stability is related to their low water activity. As yeasts and LAB naturally occur on plant materials, cereal flours carry both groups of microorganisms and competitive yeast and LAB species reach

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129

numbers above those of the adventitious microbiota upon fermentation of a flour-water mixture [57]. However, whether the type of flour mainly directs the growth of sourdough LAB species remains controversial [25, 26, 47, 89, 105, 107, 121, 123, 135]. For instance, whereas it was assumed that rice sourdough fermentation selects for Lb. spicheri [80], this LAB species cannot always be found in rice sourdoughs [123]. This has been ascribed to competitiveness of the microorganisms that are present in the sourdough ecosystem. Indeed, microbial interactions between the spontaneous microbiota and an added sourdough starter culture may lead to the dominance of autochthonous LAB species and/or strains. Among other mechanisms, competitiveness may explain the apparent prevalence of LAB species in specific sourdough preparations, such as evidenced by single reports on Lb. amylovorus in rye sourdoughs [120], Lactobacillus sakei in amaranth sourdoughs [122], and Lb. pontis in teff sourdoughs [47]. Yet, spontaneous sourdough fermentations carried out in the laboratory with flour as the sole nonsterile ingredient indicate that the type and quality (microbiological and nutritional) of the cereal flour used is indeed an important source of autochthonous LAB and yeasts occurring in the ripe sourdoughs [40, 107]. Hence, the flour plays a key role in establishing stable microbial consortia within a short time. In this context, it has been shown that laboratory sourdoughs based on wheat, rye, or spelt, backslopped daily for 10 days at 30 °C, whether or not initiated with a Lb. sanfranciscensis starter culture [as tested in the case of wheat sourdough fermentations in the study of Siragusa et al. [135]], reach an equilibrium of LAB species through a three-step fermentation process: (1) prevalence of sourdough-atypical LAB species (e.g., Enterococcus spp. and Lc. lactis subsp. lactis); (2) prevalence of sourdough-typical LAB species (e.g., species of Lactobacillus, Leuconostoc, Pediococcus, and Weissella); and (3) prevalence of highly adapted sourdough-typical LAB species (e.g., Lb. fermentum and Lb. plantarum) [107–109, 135]. Indeed, it has been shown that the LAB species Lb. fermentum (strictly heterofermentative) and Lb. plantarum (facultatively heterofermentative) dominate several sourdough fermentation processes, irrespective of the type of flour or the addition of starter cultures that are not robust enough [47, 108, 123, 136–138, 142, 172, 174, 175]. Concerning yeasts, C. glabrata and W. anomalus prevail during laboratory sourdough fermentations [40]. Further, it has been shown that previous introduction of flour into the bakery environment helps to build up a so-called house microbiota that serves as an important inoculum for subsequent bakery sourdough fermentations [155]. Indeed, LAB strains adapted to the sourdough and bakery environment (apparatus, air, etc.), which have been shown to be genetically indistinguishable, may be repetitively introduced in consecutive sourdough batches during backslopping. The widespread use of bakers’ yeast may be responsible for the prevalence of S. cerevisiae in bakery sourdoughs [26, 29, 31, 34–36, 39, 40]. However, there are also indications through reliable molecular data of a large strain diversity of S. cerevisiae in single sourdoughs, that suggest an autochthonous wheat flour origin of this yeast species in sourdough too [27, 30, 34]. Supportive of an autochthonous origin of S. cerevisiae is also the presence of this species in rye flour [29]. Yet, during laboratory fermentations with flour as the sole nonsterile ingredient and without added bakers’ yeast, other species such as C. glabrata and W. anomalus

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emerge [40]. Anyway, the direct environment is another important source of (accidental) contamination of the flour by LAB and yeasts. Consequently, hygienic conditions in the sourdough and bakery environments will play a role as well. Finally, microorganisms occurring on cereals and subsequently in sourdoughs may be of intestinal origin, due to fertilization practices on the grain fields, mouse feces or insects in the flour mills, or fecal contamination of the sourdough production environment [70, 175–178]. It may explain the opportunistic presence of Lactobacillus acidophilus, Lactobacillus johnsonii, Lb. reuteri, and Lb. rossiae, which are common gastrointestinal inhabitants.

5.2.3

Influence of Technology

Besides the cereals and other dough ingredients, which are mainly responsible as the source of metabolic activity in the form of flour enzymes and endogenous microorganisms, specific technological process parameters determine the species diversity, number, and metabolic activity of the microorganisms (whether or not added) present in the stable, ripe sourdough. These process parameters include chemical composition and coarseness of the flour, leavening and storage temperature, fermentation time, pH, redox potential, dough yield, refreshment time and number of propagation steps, and interactions between the microorganisms [57, 102, 164, 165, 179]. Different types of sourdough exist, on the basis of the processing conditions and/ or technology used for production, with a specific microbiota occurring in each type [57, 180]. Type I or traditional sourdoughs are manufactured by continuous, (daily) backslopping, at ambient temperature (